plant tissue culture- all notes

 HAPPIENESS OF LIFE
PLANT TISSUE CULTURE -BT-124

Essay on Plant Tissue Culture Contents:

 the Definition of Plant Tissue Culture.
 the History of Plant Tissue Culture.
 the Basic Requirements of Plant Tissue Culture.
 the General Techniques of Plant Tissue Culture.
 the Basic Aspects of Plant Tissue Culture.
 the Cellular Totipotency.
 the Differentiation.
 the Methods in Plant Tissue Culture.
 the Applications of Plant Tissue Culture.
 the Morphogenesis.
 the Subculture or Secondary Cell Culture.
 the Soma-Clonal Variation.
 the Somatic Hybrids and Cybrids.
 the Micro-Propagation.
 the Artificial Seed.
 the Cryopreservation.

Definition of Plant Tissue Culture:
Plant tissue culture has a great significance in plant biotechnology specially in
the crop improvement programmes. 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. However, in general, the
tissue culture includes the term tissue culture as well as cell culture, organ
culture and suspension culture also.
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.
Related Terms:
Tissue Culture:
The in-vitro culture of the tissue e.g. Callus culture
Cell Culture:
Denotes the in-vitro culture of single or a few cells.
Organ Culture:
This term is used for in-vitro culturing of organs like embryo, root or shoot
apices.
Suspension Culture:
Defined as the culture of cell and cell aggregates suspended in a liquid
medium.
Ex plant:
The excised piece of differentiated tissue or the organ which is used for culture
is called as explant. e.g., embryos, young leaf, bud, etc.
Callus:
The undifferentiated mass of cells is referred to as callus. The cells of callus are
meristematic in nature.
History of Plant Tissue Culture:
G. Haberlandt, a German botanist, in 1902 cultured fully differentiated plant
cells isolated from different plants. This was the very first step for the
beginning of plant cell and tissue culture. Further contributions were made by
the Cell Doctrine which admitted that a cell is capable of showing totipotency.
With the identification of a variety of chemicals like cytokinin, auxin, other
hormones, vitamins, etc. and their role in affecting cell division and
differentiation, the methods of plant tissue culture developed in a proper
manner. 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 multiplication of
cell also provided an extra aid for the development of in-vitro culture methods
of plants.

Basic Requirements and Techniques of Plant Tissue Culture:
The main requirements of plant tissue culture are:
(1) Laboratory Organisation
(2) Culture Media
(3) Aseptic Conditions
1. 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.

2. 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.
(ii) 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).
(iii) Carbon and Energy Source:
Most preferred carbon source is Sucrose. Others include lactose, maltose,
galactose, raffinose, cellobiose, etc.
(iv) 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.

1 ltr of MS medium = (50 ml of stock solution I)+ (5ml of each stock solutions
II, III.IV)
3. 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:
i. Sterilization of the culture vessels using detergents, autoclaves, etc.
ii. Sterilization of instruments like forceps, needles etc. by flame sterilization.

iii. Sterilization of culture medium using filter sterilization or autoclaving
methods.
iv. 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.
4. General Technique of Plant Tissue Culture:
General technique of plant cell, tissue and organ culture is almost the same
with a little variation for different plant materials. There are certain basic steps
for the regeneration of a complete plant from an explant cultured on the
nutrient medium (Fig. 1).
These basic steps for in-vitro culturing of plants are:

(a) Selection and Sterilisation of Explant:
Suitable explant is selected and is then excised from the donor plant. Explant is
then sterilized using disinfectants.
(b) Preparation and Sterilisation 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.
(c) Inoculation:
Sterilized explant is inoculated (transferred) on the culture medium under
aseptic conditions.
(d) Incubation:
Cultures are then incubated in the culture room where appropriate conditions
of light, temperature and humidity are provided for successful culturing.
(e) Sub culturing:
Cultured cells are transferred to a fresh nutrient medium to obtain the
plantlets.

(f) 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.
Equipment in Tissue Culture Lab:

5. Basic Aspects of Plant Tissue Culture:
In plant tissue culture technique, an explant is taken, it is cultured on a
nutrient medium under certain conditions and finally we obtain a whole new
plant. How does it happen?
The answer to this question lies in the inherent capacities of plant cells that are
differentiation and cellular totipotency.
6. 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 tells 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.

Dedifferentiation and re-differentiation are the two inherent phenomena
involved in the cellular totipotency. Regarding this, it is clear that the cell
differentiation is the basic event for development of plants and it is also
referred to as cyto-differentiation.
To express its totipotency, a differentiated cell first undergoes the
phenomenon of dedifferentiation and then undergoes the re-differentiation
phenomenon (Fig. 3). Usually the dedifferentiation of the explant leads to the
formation of a callus. However, the embryonic explants, sometimes, result in
the differentiation of roots or shoots without an intermediary callus state.

Thus, from the above account it is clear that unlike animals (in which
differentiation is irreversible usually), the plants have such a quality that even
highly mature and differentiated cells have an ability to revert back to
meristematic state. The property of totipotency of plant cells indicate that even
the undifferentiated cells of a callus carry the essential genetic information
required for regeneration of a whole plant.
It is also clear that all the genes responsible for dedifferentiation or re-
differentiation are present within the individual cells and they become active
for expression under adequate culture conditions. As totipotent cells are the
basis of whole plant tissue culture techniques, so, by the exploitation of this
potential of plant cells, biotechnologists are trying to improve the crop plants
and other commercially important plants.
Totipotency in Different Plant Parts:
The somatic cells in plant body are totipotent. It is to be noted here that only
the living plant cells have the ability to regenerate and the dead cells which
lack cytoplasm and nucleus (tracheid’s, vessel elements, etc.) are not totipotent
at all.

Different plant parts have different totipotent abilities. For example, in tobacco
plant, the type of bud formed by in-vitro culture of the epidermis of different
regions of the plant are different in their form.
Another example to add here may be given about the totipotency of crown-gall
cells which have the capacity to grow as an un-organised mass of cells under
normal conditions, however whole plants can be recovered from them in
culture. Thus, it is clear that totipotency is not similar in all plant parts.
Applications of Totipotency:
Cellular totipotency of plants cells has proved to be a boon to mankind as it is
the basis of plant tissue culture. The plant tissue culture exploits this unique
property of plant-cells to attain commercial benefits.
Various applications of cellular totipotency are:
i. It has potential applications in the crop plant improvement.
ii. Micro-propagation of commercially important plants.
iii. Production of artificial or synthetic seeds.
iv. It helps in conservation of germplasm (genetic resources).
v. This ability is utilized for haploid productions.
vi. Applied in producing somatic hybrids and cybrids.
vii. Helps in cultivation of those plants whose seeds are very minute and
difficult to germinate.
viii. Also helps to study the cytological and histological differentiations.
ix. For high scale and efficient production of secondary metabolites.
x. The genotypic modifications can also be possible.
7. Differentiation:
While studying totipotency, it is stated that the dedifferentiation and
redifferentiation processes result in the differentiated plant organs, finally
producing a whole plant. In case of plants, the differentiation is reversible but
in animals, it is irreversible.

The term differentiation describes the development of different cell types as
well as the development of organised structures like roots, shoots, buds, etc.,
from cultured cells or tissue.
Differentiation may also be defined in simple words as the development
change of a cell which leads to its performance of specialised function.
However, normally morphological characteristics. For example, differentiation
accounts for the origin of different types of cells, tissues and organs during the
formation of a complete multicellular organism (or an organ) from a single-
celled zygote.
Actually, the development of an adult organism starting from a single cell
occurs as a result of the combined functioning of cell division and cell
differentiation. Various techniques of tissue culture provide not only a scope of
studying the factors governing totipotency of cells but also serves for the
investigation of patterns and factors controlling the differentiation.
Types of Differentiation:
As stated earlier also, the plant cells have a tendency to remain in a quiescent
stage which may be reverted to the meristematic stage. This process is termed
as dedifferentiation and as a result of this, a homogeneous undifferentiated
mass of tissue i.e., callus is formed. There callus cells then differentiate into
different types of cells or an organ or an embryo.
On this basis, the differentiation may be of the following types:
(a) Cytodifferentiation
(b) Organ Differentiation
(c) Embryo Genic Differentiation
a. Cytodifferentiation:
The differentiation of the cells is an important event of the development of
plants. The differentiation of different types of cells from the cultured cells is
known as cytodifferentiation. When an undifferentiated callus re-differentiates
into whole plant, it first undergoes cytodifferentiation.
Amongst different cytodifferentiations, the differentiation into vascular tissues
has received maximum attention. However, it is important here to mention
that the cells of mature xylem elements and phloem cells cannot be re-
differentiated or cannot be reverted back to the meristematic state due to lack
of cytoplasm in them.

Although, in initial stages of their development, they can be reverted to
meristematic cells. Xylogenesis is the differentiation of parenchymatous cells
(of callus) into xylem-like cells of vascular plants. Phloem differentiation is the
formation of phloem-cells from parenchyma in culture.
Factors affecting cytodifferentiation:
(i) Physical factors like light, temperature and pH are effective at optimum
levels.
(ii) Chemical factors.
a. Low Nitrogen content increases vascularization
b. High Ca
++ ions stimulates the formation of tracheid’s and sieve tubes.
c. Sucrose in high concentration results in pronounced xylem differentiation.
(iii) Hormones:
Some hormones play important role in cytodifferentiation.
These are:
a. Auxin plays major role in vascularization.
b. Cytokinin promotes cytodifferentiation.
c. Gibberellins along with auxins promote it.
d. Abscisic acid inhibits it usually.
b. Organ Differentiation:
It is synonymous to organogenesis or organogenic differentiation. It refers to
the development or regeneration of a complete organised structure (or whole
plant) from the cultured cells/tissues (Fig. 4).

Organogenesis literally means the birth of organ or the formation of organ. It
may occur either by shoot bud differentiation or by the formation of root.
Organogenesis commences with the stimulus produced by the components of
culture medium, the substances initially present in the original explants and
also by the compounds produced during culturing.
Among different organs, which can be induced in plant tissue culture are
included the roots, shoots, flower buds and leaves. Regenerations into flower
buds and leaves occur in a very low frequency.
However, the roots and shoot bud regenerations are quite frequent. Out of all
these types of organogenic differentiation, only the shoot bud differentiation
can give rise to the complete plantlets therefore, it is of great importance in
tissue culture practices.
The initiation of roots is termed as rhizogenesis while the initiation of shoots is
called as caulogenesis and these two phenomena are affected by alterations in
the auxin : cytokinin ratio in the nutrient medium. A group of meristematic
cells called as meristemoids is the site of organogenesis in callus. Such
meristemoids are capable of producing either a root or a shoot.

Organogenesis may occur either through callus formation or through the direct
formation of adventitious organs (like adventitious shoot). Latter mode of
organogenesis does not involve the intervening callus phase.
Shoot bud differentiation was first of all demonstrated by White (1939).
Further, in 1944, Skoog indicated that organogenesis could be chemically
controlled. Shoot bud differentiation refers to the formation of shoot buds
from the cultured cells by providing appropriate culture conditions and
nutrient medium. The chemical and physical factors required for shoot bud
differentiation vary for explants from different plant species.
Factors affecting organogenesis:
(i) Auxin: Cytokinin ratio in medium is an important factor affecting
root/shoot bud differentiation in most plants.
(ii) Usually Gibberellic acid inhibits organogenesis.
(iii) Physiological state and size of explant play important role in organ
differentiation.
(iv) Genotype of the donor plant plays a crucial role.
(iv) Physical factors like light, temperature, moisture, etc., play effective role in
organogenesis.
c. Embryo Genic Differentiation:
The embryos formed from the somatic cells of plant in culture under in-vitro
conditions are called as somatic embryos. When the somatic cells of plant
organs result into the regeneration into embryos, then the process is called as
somatic embryogenesis or embryo genic differentiation or embryogenesis (Fig.
5).

Somatic embryos are also referred to as embryoids, and they can be obtained
either indirectly (with formation of callus) or directly from the explant without
intervening callus formation. However, direct embryogenesis is not a normal
process because the medium requirement for this is complex.
Somatic embryogenesis under in-vitro conditions was first of all observed by
Steward et. al. (1958) in carrot (Daucus carota). Thereafter, somatic embryoids
have been induced in many plants namely Citrus, Coffea, Zea mays, etc. To
obtain embryoids, there is a requirement of two nutrient media, first for
initiation and the other medium for proper development of the embryoid.
The development of somatic embryo passes through the stages like globular,
heart-shaped, torpedo-shaped and finally giving rise to the cotyledonary stage
of somatic embryo. A somatic embryo does not have any vascular connection
with the explant or callus therefore it can be separated easily.
Somatic embryogenesis is not used very frequently for propagation of plants
because, the technique is usually difficult and also, there is a high risk of
occurrence of mutations. Another major drawback of somatic embryogenesis is

that there are greater chances of loss of regenerative capacity on repeated sub-
culturing.
Factors affecting Embryogenesis:
a. Physiological condition and type of explant.
b. Genotype of donor plant.
c. Growth regulators:
i. Auxin is essential for embryo initiation
ii. Cytokinin promotes embryogenesis
iii. Gibberellins inhibit embryo genic differentiation
iv. Abscisic Acid (ABA) suppresses it.
d. Nitrogen and Oxygen concentration
e. Physical factors like temperature and light.
8. Methods in Plant 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:
1. 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.
The method of cell culture is meritorious over other methods of culturing
because it serves as the best way to analyse and understand the cell
metabolism and effects of different chemical substances on the cellular
responses. Single cell culturing is of immense help in crop improvement
programmes through the extension of genetic engineering techniques in higher
plants.

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 (Fig. 6).

(c) Testing of cell viability done with the phase contrast microscopy or certain
special dyes.
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.
2. 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 technique for breakage of larger cell clumps (Fig. 7).

The general technique of suspension culture involves basically two types of
cultures: batch culture and continuous cultures.
A batch culture is a suspension culture in which cells grow in a finite volume of
the culture medium and as a result, medium gradually depletes. On the other
hand, a continuous suspension culture is the one which is continuously
supplied with nutrients by the inflow of fresh medium but the culture volume
is normally constant.
3. 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.
4. 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).

The apical meristem of a shoot is the portion which is lying beyond the
youngest leaf primordium. 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.
5. 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.
6. Haploid Production:
Haploid plants are those which contain half the number of chromosomes
(denoted by n). Haploids can be exploited for benefits in the studies related to
experimental embryogenesis, cytogenetics and plant breeding. Haploids have
great significance in field of plant breeding and genetics. They are most useful
as the source of homozygous lines.
In addition, the in-vitro production of haploids also aids for induction of
genetic variabilities, disease resistance, salt tolerance, insect resistance, etc.
Presently, attention is being focused on improving the frequencies of haploid
production in their advantageous utilization for economic plant improvement.
There are two approaches for in-vitro haploid production. These
are:
(a) Androgenesis:
The technique of production of haploids through anther or microspore culture
is termed as androgenesis. It is a method par excellence for the large scale
production of haploids through tissue culture.
Androgenesis technique for haploid production is based on the in-vitro culture
of male gametophyte i.e., microspore of a plant resulting into the production of
complete plant from it. It is achieved either by another culture or by
microspore (pollen) culture.
The technique of another culture is quicker for practical purposes and is an
efficient method for haploid production.
But sometimes during another culture, the plantlets may originate from
different other parts of anther also (along with from the pollens). On the other
hand, microspore culture is free from any uncontrolled effects of the anther
wall or other tissues. Microspore culture is ideal method for studying the
mutagenic and transformation patterns (Fig. 9).

(b) Gynogenesis:
It is an alternative source of in-vitro haploid production. It refers to the
production of haploid plant from ovary culture or ovule culture. The method of
gynogenesis for haploid production has been successful, so far, in a very few
plants only, hence it is not a very popular method for in-vitro production of
haploids. Thus, androgenesis is preferred over gynogenesis.
7. Embryo Culture:
The technique of embryo culture involves the isolation and growth of an
embryo under in-vitro conditions to obtain a complete viable plant. First
success for embryo culture was made by Hannig in 1904 when he isolated and
cultured embryos of two crucifers namely Cochleria and Raphanus. Embryo
culture is used widely in the fields of agriculture, horticulture and forestry for
production of hybrid plants.
This technique allows the detailed study about the nutritional requirements of
embryos during different developmental stages. Also, it helps for identifying
the regeneration potential of embryos. Embryo culture is advantageous for in-
vitro micro propagation of plants, overcoming seed dormancy and for
production of beneficial haploid plants.

8. Endosperm Culture (Triploid Production):
Endosperm tissue is triploid therefore the plantlets originating by the culture
of endosperm are also triploid.
In majority of flowering plant families (exceptions being Orchidaceae,
Podostemaceae, Trapaceae which lack endosperm) the endosperm tissues are
present. Endosperm is formed after the double fertilization of one male
nucleus with two polar nuclei. Immature endosperm has more potential of
growth in culture especially among the cereals.
Endosperm culture has provided a novel strategy for plant breeding and
horticulture for the production of triploid plantlets. It is an easy method for
production of a large number of triploids in one step.
Moreover, it is much more convenient that the conventional techniques like
chromosome doubling by crossing tetraploids with diploids for triploid
induction. Full triploid plants of endosperm origin have been produced in a
number of plant species like Populus, Oryza sativa, Emblica officinalis, Pyrus
malus, Prunus, etc.
The triploid plants are usually seedless therefore this technique is most
beneficial for increasing the commercial value of fruits like apple, mango,
grapes, watermelon, etc. In addition to all the above described applications,
endosperm culture is helpful for studying biosynthesis and metabolism of
certain natural products also.
9. Applications of Plant Tissue Culture:
1. Germplasm conservation mainly in the form of cryopreservation of somatic
embryos or shoot apices, etc.
2. Large scale production of useful compounds and secondary metabolites by
using genetically engineered plant tissue cultures.
3. Technique of micro propagation for enhancing the rate of multiplication of
economically important plants.
4. Eradication of systemic diseases in plants and raising disease free plants.
5. Soma-clonal variations are useful sources of introduction of valuable genetic
variations in plants.

6. Helps plants in imparting resistance to antibiotics, drought, salinity,
diseases, etc.
7. Somatic hybrids and cybrids overcome species barriers and sexual
incompatibility and produce hybrid plants with desired combination of traits.
8. Embryo culture helps in overcoming seed sterility and dormancy.
9. Haploid production in culture helps to solve various problems of genetic
studies and thus aids the plant breeders for producing new varieties.
10. Production of synthetic seeds via somatic embryo differentiation for
commercially important plants helps to achieve increased agricultural
production.
11. Large scale production of biomass energy.
12. Plant tissue culture aids in producing the genetically transformed plants.
13. Early flowering can be induced by in-vitro culturing of plants so as to attain
commercial benefits.
14. Triploids as well as polyploid plants can also be produced by tissue culture
techniques for uses in plant breeding, horticulture and forestry.
15. Seedless fruits and vegetables can be produced by following the endosperm
culture method which add to their commercial values.
16. Increased Nitrogen fixation ability can be achieved through association of
tissue culture techniques with genetic engineering.
17. Callus cultures are useful in plant pathology as they act as an effective tool
in the study of mechanism of disease resistance and susceptibility.
18. Different tissue culture techniques help us to study various biosynthetic
processes, physiological changes and cytogenetic changes.
10. Morphogenesis:
Literal meaning of the term morphogenesis is origin of form. It includes all the
activities which are involved in the formation of a complete individual starting
from the cells/tissues in culture. Various internal as well as external factors
affect the morphogenic potential of the cells.

The study of such factors and their effect on the regeneration of cells
constitutes the morphogenesis. The appearance of the plants may be altered by
the changes in growth environment. The causal factors of such changes may be
studied properly by morphogenesis.
Morphogensis may also be termed as developmental morphology and it may be
defined as “the branch of biology that deals with the causes and
activities during the organization of a complete individual
plant.” Another definition of morphogenesis can be given as the study of
various factors which affect the organic form and describe the growth patterns
of the individual.
All those anatomical and physiological events which are involved in the growth
and development of an organism are included in the morphogenetic studies.
Differences in the morphogenetic phenomena lead to the differences in the
form of characteristic organs or structures. In simple words, morphogenesis
may be described as the developmental pathways in differentiation as a result
of which the recognizable tissues are formed.
Cell culturing has blossomed into such a technology which is capable of solving
a number of problems with an impact on morphogenesis of plants. This is so
because, through the elegant system of plant cell/tissue culture, it is possible to
have an effective control of exogenous factors which affect the differentiation
starting with the callus or a single cell or tissue.
Factors of Morphogenesis:
There are many such factors which influence the morphogenic potential of
cells by affecting important developmental activities. For e.g.,
i. Several experiments have shown that certain growth factors (like cytokinin)
induce the somatic embryos in culture by changing the cell polarity.
ii. Some chemical factors promote asymmetric divisions in the cultured cells
thus resulting in difference in form.
iii. Different concentrations of 2, 4-D, auxins, etc. alter the development of
organ in culture by causing the altered cell elongation or rate of cell division.
iv. With the increase in culture duration, the organogenic differentiation shows
decline. All such examples show that different factors have different type of
effect on different developmental activities. Such dynamic and causal aspects
of organic forms are studied in morphogenesis.

A list of the factors having great importance in deciding the
morphogenic pattern is given below:
i. Physical factors like light, temperature, pH.
ii. Chemical factors like C/N ratio, oxygen content, carbohydrates (sugar)
concentrations, etc.
iii. Growth hormones like Auxin, Cytokinin, Gibberellins, ABA, etc.
iv. Water
v. Culture duration
vi. Mechanical strains, pressures, bending, etc., may cause changes in plane of
cell division, thus may cause difference in morphogenic potential.
vii. The regenerability of explant may be affected by factors like the organ from
which it is taken, the physiological state of the explant, its size, etc.
viii. Electrical stimulation.
ix. Changes in gene expression in cells in culture.
x. Genotype and status of nucleic acids in different cells.
xi. Repeated subculture of a totipotent callus may also result in reduction or
complete loss of morphogenetic potential.
Role of Morphogenesis:
As described above, morphogenesis includes all those events which occur
during the growth and development of an organism. Thus, it is very important
for providing a satisfactory explanation of various biological growth patterns.
It also aims on studying and correlating different environmental factors with
changes in the morphogenetic patterns and/or potential.
Major contributions for morphogenetic studies in India have been provided by
Department of Botany, University of Delhi. Numerous experiments on a wide
variety of subjects were performed by the department to study the
morphogenesis by utilizing tissue culture techniques.
De-novo organ initiation and formation under aseptic culture form a
fascinating subject to study the effect of different factors on the morphogenetic
patterns.

Different objectives of morphogenesis are summarized below:
i. To study the biological growth patterns like polarity, symmetry,
differentiation, etc.
ii. To provide an explanation to all such growth patterns.
iii. To relate morphogenesis with other major biological sub sciences.
iv. Advantageous utilization of tissue culture techniques to study
morphogenetic patterns.
v. Determining interacting influence of growth hormones in shoots or root
regeneration.
vi. To study the effects of repeated sub-culturing on the regeneration potential
of a totipotent callus.
vii. To analyse the growth regulation in differentiation of plant species by trial
and error.
viii. Study of effect of osmotic inhibition using high levels of sucrose during
shoot formation.
ix. Study of role of light and temperature in flowering.
x. Exploring the correlation between nuclear state and potential for
differentiation and regeneration.
11. Subculture or Secondary Cell Culture:
The process of transferring the cultured cells in a fresh nutrient medium is
called as sub-culturing, and the cell cultures which are sub-cultured (i.e.,
inoculated in a separate medium) are called as subculture or secondary cell
cultures or secondary cell lines.
It is important to subculture the organ and tissues to fresh medium to avoid
the condition of nutrition depletion and drying of medium. It is possible to
maintain the plant cell and tissue cultures for indefinitely long time durations
if they are regularly subculture in a serial manner (Fig. 10).

12. Soma Clonal Variation:
Soma clonal variations may be defined as those variations which occur in the
cultured cells/tissues or plants regenerated from such cells in-vitro. Soma
clonal variations are usually heritable for qualitative as well as quantitative
characters of plants. Soma clonal variants have proved as an alternate tool to
plant breeding for production of improved varieties of plants.
Gene mutations and changes in the structure, number of chromosomes are the
main causes of production of soma clonal variants. A number of new varieties
of cereals, oil seeds, fruits, tomatoes, etc., possessing disease resistance, better
quality, better yield etc., have been generated through soma clonal variations.
Some of those crop species are potato, tomato, oats, wheat, rice, maize, datura,
carrot, soybean, etc.
In simple words, the variability which is generated by the use of a tissue
culture cycle is termed as soma clonal variation. Soma clones is a general term
which is used to describe those plants which have been derived from any type
of somatic cell cultures.
13. Somatic Hybrids and Cybrids:
Somatic hybridization may be described as the production of hybrid cells by
the fusion of protoplasts of somatic cells derived from two different plant
species/varieties. It is immensely helpful for generating new and improved
hybrid varieties of plant that may have characters of a completely different
species.
For example, ‘Pomato’ is a somatic hybrid which is produced by the fusion of
protoplast of somatic cells from potato and tomato which are totally different
species. A cybrid is a cytoplasmically hybrid cell which has the cytoplasm of
both fusing cells but nucleus of only one fusing cell. The process of production
of a cybrid is called cybridisation.
Steps involved in somatic hybridization/cybridization (Fig. II) are
given below:

a. Protoplast isolation from parent plant using any mechanical or enzymatic
method.
b. Fusion of isolated protoplasts derived from two different parents either by
utilizing chemical fusogens (like NaNO3, Polyethylene Glycol or high pH-high
Ca
++ treatment) or by the electro-fusion method.
c. Selection of the hybrid cells is done after fusion process.
d. The selected somatic hybrids/cybrids are then verified for hybridity. This is
done to check whether the hybrid is carrying the desired characteristics of both
parents or not.
e. On successful verification and characterization, the somatic hybrids or
cybrids are cultured for regenerating into the plantlets with desired characters.
Somatic hybridization overcomes the sexual incompatibility barriers and it
enables to produce interspecific as well as inter-generic crosses in plants. It
helps in imparting disease-resistances and improving the quality characters in
plants. It is an immensely beneficial tool for study of cytoplasmic genes and
their expressions.

14. Micro-Propagation:
Tissue culture helps in the rapid propagation of plants by the technique of
micro-propagation or clonal propagation in-vitro. The asexually produced
progeny of a cell or individual is called as clone and the clones have an
identical genotype.
Micro propagation is the technique of in-vitro production of the clones of
plants i.e., it produces the progeny plants which have an identical genotype as
their parents, by cell, tissue or organ culture (Fig. 12). It helps in the
production of plants in large number starting from a single individual. It serves
as an alternate method to conventional vegetative propagation methods.

Micro propagation may be achieved by shoot tips, axillary buds, adventitious
buds, bulbs or somatic embryos. A number of plant species are being clonally
propagated in vitro, specially the commercially valuable plants like orchids and
forest trees.
Some of the important plants that have been micro propagated on
large scales are:
Orchids like Cymbidium, Dendrobium, Aranda, Vanda, Odontoma, Vanilla,
etc.
Forest trees like Tectona grandis, Biota, Cedrus deodara, Eucalyptus, Picea,
Pinus, etc.
The technique of micro propagation generally involves four stages. Each of
these stage has its own requirements.
The stages in general technique of micro-propagation are described
below:
Stage I. Initiation:

This stage also involves the preparatory process for achieving better
establishment of aseptic cultures of explant. Suitable explant is selected from
the mother plant. Then, the explant is sterilized and transferred to the nutrient
medium for culture.
State II. Multiplication:
This is the most important stage of micro propagation. In this stage, there
occurs the proliferation or multiplication of shoots (or embryoids) from the
explant on medium. It occurs either by the formation of an intermediary callus
or by induction of adventitious buds directly from the explant.
Stage III. Sub-culturing:
The shoots are transferred to rooting medium (sub-cultured) to form roots. As
a result, complete plantlets are obtained.
Stage IV. Transplantation:
In this stage, the regenerated plantlets are transferred out of culture. These are
grown in pots followed by field trials.
Advantages of Micro Propagation:
1. Rapid multiplication of disease free plants.
2. Rapid multiplication of commercially important plants.
3. Maintenance of genetic uniformity.
4. Technique does not depend on seasons and is capable of producing plants all
round the year.
5. Technique is valuable in cases where only limited explant is available.
Limitations of Micro Propagation:
1. The technique is costly.
2. It requires proper skill.
3. Many tree crops, including gymnosperms, cannot be multiplied by clonal
propagation.
4. Clonal propagation in some cases may lead to the formation of off-types
rather than clones, after many generations.

5. If culture is contaminated, then the pathogen gets multiplied to very high
levels and becomes difficult to handle.
15. Artificial Seed:
These are also called as synthetic seeds. These are living seed like structures
which are capable of giving rise to plants when sown in the field. An artificial
seed is made of a somatic embryo (S.E.) encapsulated with a protective layer of
a gel which protects it from desiccation or microbial attack (Fig. 13).

16. Cryopreservation:
It means preservation at ultralow temperature:
This technique is used mainly for long term storage of germplasm and thus
helps in conservation of nature also. Plant tissues and organs are
cryopreserved usually in liquid Nitrogen which has a temperature of 196°C.
Cryopreservation technique has proved to be one of the most reliable methods
for long term storage and preservation of plant germplasm in the form of
pollens, shoot-tips, embryos, callus, protoplasts, etc.
Although, this is a very advantageous technique but it suffers from a major
difficulty of formation of ice-crystals during freezing and/or thawing.
These ice-crystals may cause damage to the preserved material. To prevent the
formation of ice-crystals during cryopreservation, some special chemicals are
used which are called as cryoprotectants. A few common cryoprotectants are
glycol, sucrose, proline, Dimethyl Sulfoxide (DMSO), Polyethyleneglycol
(PEG), etc..
Vascular differentitation:
Vascular tissues, xylem and phloem, are differentiated from meristematic cells,
procambium, and vascular cambium. Auxin and cytokinin have been
considered essential for vascular tissue differentiation; this is supported by
recent molecular and genetic analyses. Xylogenesis has long been used as a

model for study of cell differentiation, and many genes involved in late stages
of tracheary element formation have been characterized. A number of mutants
affecting vascular differentiation and pattern formation have been isolated in
Arabidopsis. Studies of some of these mutants have suggested that vascular
tissue organization within the bundles and vascular pattern formation at the
organ level are regulated by positional information.
Totipotency?
Totipotency is the genetic potential of a plant cell to produce the entire plant.
In other words, totipotency is the cell characteristic in which the potential for
forming all the cell types in the adult organism is retained.
Expression of Totipotency in Culture:
The basis of tissue culture is to grow large number of cells in a sterile
controlled environment. The cells are obtained from stem, root or other plant
parts and are allowed to grow in culture medium containing mineral nutrients,
vitamins and hormones to encourage cell division and growth. As a result, the
cells in culture will produce an unorganised proliferative mass of cells which is
known as callus tissue.
The cells that comprise the callus mass are totipotent. Thus a callus tissue may
be in a broader sense totipotent, i.e., it may be able to regenerated back to
normal plant given certain manipulations of the medium and the cultural en-
vironment. Truly speaking, totipotency of the cell is manifested through the
process of differentiation and the hormones in this process play the major role
than any other manipulations.
In the fifties, F. Skoog and C.O. Miller of U.S.A. discovered a new plant growth
hormone kinetin from herring sperm DN A. With a correct concentration ratio
of auxin and cytokinin in tobacco cultures, Skoog was able to demonstrate the
role of kinetin in organogenesis. When the ratio of kinetin to auxin was higher,
only shoot developed. This is known as caulogenesis. But when the ratio was
lower, only roots were formed. This is known as rhizogenesis.
Around the same period, F. C. Steward and his colleagues at Cornell University
of USA, devised a method for growing carrot tissue by excising small disc, from
the secondary phloem region of carrot root and placing them in a moving
liquid medium under aseptic conditions. In presence of coconut milk in the
medium, the phloem tissue began to the grow actively.

In moving liquid medium some single cells and small groups of cells were
loosened from the surface of growing tissue. When these isolated cells were
grown separately it was found that some single cells developed somatic
embryos or embryoids by a process that occurs in normal zygotic embryo
It is also observed in some experiment that cells of some callus mass frequently
differentiate into vascular elements such as xylem and phloem without forming
any plant organs or embryoids. This process is known as histogenesis or Cyto-
differentiation. Thus the totipotent cells may express themselves in different
way on the basis of differentiation process and manipulation.
Where the totipotent cells are partially expressed or not expressed, it is
obvious that the limitation on its capacity for development must be imposed by
the microenvironments. The totipotency of cells in the callus tissue may be re-
tained for a longer period through several subcultures.
Practically, it is observed that the ex- plant first forms the callus tissue in the
callus inducing medium and such callus tissue is maintained through some
subcultures. After then it is generally transferred to another medium which is
expected to be favourable for the expression of totipotent cells. Actually, the
regeneration medium is standardized by trial and error method.
In more or less suitable medium, the totipotent cells of the callus tissue give
rise to meristematic nodules or meristemoids by repeated cell division. This
may subsequently give rise to vascular differentiation or it may form a
primordium capable of giving rise to a shoot or root. Sometimes the totipotent
cell may produce embryoids through sequential stages of development such as
globular stage, heart shaped stage and torpedo stage etc.
After prolonged culture, it has been observed that calluses in some species (e.g.
Ntcotiana tabacum, Citrus aurantifolia etc.) maybe- come habituated. This
means that they are now able to grow on a standard maintenance medium
which is devoid of growth hormones. The cells of habituated callus also remain
totipotent and are capable to regenerate a plant without any major
manipulation.

A typical crown gall tumour cell has the capacity for unlimited growth
independent of exogenous hormones. It shows totally lack of organ genic
differentiation. So such tissue is considered to have permanently lost the
totipotentiality of the parent cells.
In some plant species, the crown gall bacterium (Agrobactenum tumefaciens)
induces a special type of tumour, called teratomas, the cells of which possess
the capacity to differentiate shoot buds and leaves when they are grown in
culture for unlimited periods. Thus it is clear that the mode of expression of
totipotency of plant cell in culture varies from plant to plant and also helps us
to understand the process of differentiation in vitro.
Importance of Totipotency in Plant Science:
The ultimate objective in plant protoplast, cell and tissue culture is the
reconstruction of plants from the totipotent cell. Although the process of
differentiation is still mysterious in general, the expression of totipotent cell in
culture has provided a lot of information’s.
On the other hand, the totipotentiality of somatic cells has been exploited in
vegetative propagation of many economical, medicinal as well as agriculturally
important plant species. Therefore, from fundamental to applied aspect of
plant biology, cellular totipotency is highly important.
Recent trends of plant tissue culture include genetic modification of plants,
production of homozygous diploid plants through haploid cell culture, somatic
hybridization, mutation etc. The success of all these studies depends upon the
expression of totipotency. In many cases, successful and exciting results have
been obtained.
Plant breeders, horticulturists and commercial plant growers are now more
interested in plant tissue culture only for the exploitation of totipotent cells in
culture according to their desirable requirement. Totipotent cells within a bit
of callus tissue can be stored in liquid nitrogen for a long period. Therefore, for

germplasm preservation of endangered plant species, totipotency can be
utilized successfully.

CULTURE MEDIA:
Growth and morphogenesis of plant tissues in vitro are largely governed by the
composition of the culture media. Although the basic requirements for
culturing plant tissue is same but in practice there are some specific nutritional
requirements for promoting optimal growth from different kinds of explants in
case of different plant species.
Components of Media:

The principal components of most plant tissue culture media are inorganic
nutrients (macronutrients and micronutrients), carbon sources, organic
supplements, growth regulators, vitamins, amino acids and gelling agent for

solid or semisolid media. The various culture media formulated for plant tissue
culture are Murashige and Skoog’s medium, Gamborg’s medium, White’s
medium, etc. which differ (Table 16.1) mainly in quantity of the components.
(i) Inorganic Nutrients:
A variety of mineral elements (salts) supply the macro- and micronutrients
required in the life of a plant. Elements required in concentration greater than
0.5 m mol/1 referred to as macronutrients and those less than 0.05 m mol/1 as
micronutrients.
The macronutrients include six major elements like Nitrogen (N), Phosphorus
(P), Potassium (K), Calcium (Ca), Magnesium (Mg) and Sulphur (S) which are
present as salts in various media. The micronutrients are iron (Fe), Manganese
(Mn), Zinc (Zn), Boron (B), Copper (Cu), Molybdenum (Mo). Among these
iron is used in the medium as chelated form with EDTA (ethylene-diamino-
tetra-acetic acid).
(ii) Carbon and Energy Source:
Plant cells and tissues in the culture medium lack autotrophic ability and
therefore need external carbon for energy. The most preferred carbon source in
tissue culture media is sucrose; glucose supports equally for good growth while
fructose is less efficient.
(iii) Vitamins:
The plant cells and tissues are capable of synthesizing different vitamins but in
in vitro condition they are being produced at sub-optimal level. Hence it is
necessary to supplement the media with required vitamins such as Thiamine
(B1), Nicotinic acid (B3), Pyridoxine (B6), Pantothenic acid (B5) and myoinositol.
Except these other vitamins like Biotin, Folic acid, Ascorbic acid are also used
in some media.
(iv) Amino Acids:
In vitro grown plant cells or tissues are capable of synthesizing amino acids but
the addition of amino acids to media is important for stimulating cell growth
and protoplast culture and for establishment of cell lines. Glutamine,
asparagine, glycine, arginine, cysteine are the commonly used amino acids.
(v) Growth Regulators:
Three broad classes of growth regulators mainly auxins, cytokinins and
gibberellins are used in tissue culture. The growth, differentiation and
organogenesis of tissues become feasible only on the addition of one or more of
these classes of hormones to a medium.

Various kinds of auxins like IAA (Indole-3-Acetic Acid), NAA (Naphthalene
Acetic Acid), 2, 4-D (2, 4-dichlorophenoxy acetic acid) are used to induce cell
division, elongation of stem, internodes and rooting.
Cytokinins like BAP (Benzyl amino purine), Kinetin (Furfuryl amino purine),
Zeatin or 2iP (Isopentynyl adenine) are used which are mainly concerned with
cell division, modification of apical dominance and shoot differentiation in
tissue culture.
The ratio of auxin and cytokinin is important with respect to morphogenesis in
the culture system. For embryogenesis, callus initiation and root initiation the
requisite ratio of auxin to cytokinin is high, while the reverse leads to
organogenesis and shoot proliferation. Gibberellins (GA3) are occasionally used
growth regulators to induce plantlet formation from adventive embryos
developed in culture.
(vi) Other Organic Supplements:
Culture media are often supplemented with variety of organic extracts which
have the constituents of an undefined nature e.g. casein hydrolysate, coconut
milk, malt extracts and fruit juice.
(vii) Activated Charcoal:
The addition of activated charcoal to culture media is reported to stimulate
growth and differentiation by reducing toxicity by removing toxic substances
(e.g. Phenols).
(viii) Antibiotics:
For transformation experiments addition of antibiotics to culture media is
required to prevent the growth of Agrobacterium which retards the cell or
tissue growth.
(ix) Gelling Agent:
Gelling or solidifying agents are commonly used for preparing semisolid or
solid tissue culture media to provide solid surface area for growth.
Agar (a polysaccharide obtained from seaweeds), Gelatin (commercially avai-
lable as Phytagel, Alginate or Gelrite) are commonly used solidifying agents at
a concentration of 0.8-1.0% (which are not any kind of nutrient), depending on
the type of tissue and the species of plant and culture methods.
Media Preparation:
For media preparation it is convenient to use stock solutions of major salts
(20X), minor salts (200X), organic supplements (200X), growth regulator (1 m

mol or 10 m mol). For final media preparation the stock solutions are mixed
together in appropriate quantities. All the stock solutions are stored in proper
plastic or glass containers at low temperature.
After preparation of media the pH is adjusted at 5.5-6.0 and agar is mixed
whenever it is required. The media is poured into the desired type of culture
vessels (culture tubes, conical flasks, petriplates, etc.), properly plugged with
non-absorbent cotton and autoclaved.
PLANT TISSUE CULTURE MEDIA:
Basically, the plant tissue culture media should contain the same nutrients as
required by the whole plant. It may be noted that plants in nature can
synthesize their own food material. However, plants growing in vitro are
mainly heterotrophic i.e. they cannot synthesize their own food.
Composition of Media:
The composition of the culture media is primarily dependent on two
parameters:
1. The particular species of the plant.
2. The type of material used for culture i.e. cells, tissues, organs, protoplasts.
Thus, the composition of a medium is formulated considering the specific
requirements of a given culture system. The media used may be solid (solid
medium) or liquid (liquid medium) in nature. The selection of solid or liquid
medium is dependent on the better response of a culture.
Major Types of Media:
The composition of the most commonly used tissue culture media is given in
Table 43.1, and briefly described below.

White’s medium:
This is one of the earliest plant tissue culture media developed for root culture.
MS medium:
Murashige and Skoog (MS) originally formulated a medium to induce
organogenesis, and regeneration of plants in cultured tissues. These days, MS
medium is widely used for many types of culture systems.
B5 medium:

Developed by Gamborg, B5 medium was originally designed for cell
suspension and callus cultures. At present with certain modifications, this
medium is used for protoplast culture.
N6 medium:
Chu formulated this medium and it is used for cereal anther culture, besides
other tissue cultures.
Nitsch’s medium:
This medium was developed by Nitsch and Nitsch and frequently used for
anther cultures. Among the media referred above, MS medium is most
frequently used in plant tissue culture work due to its success with several
plant species and culture systems.
Synthetic and natural media:
When a medium is composed of chemically defined components, it is referred
to as a synthetic medium. On the other hand, if a medium contains chemically
undefined compounds (e.g., vegetable extract, fruit juice, plant extract), it is
regarded as a natural medium. Synthetic media have almost replaced the
natural media for tissue culture.
Expression of concentrations in media:
The concentrations of inorganic and organic constituents in culture media are
usually expressed as mass values (mg/l or ppm or mg I
-1). However, as per the
recommendations of the International Association of Plant Physiology, the
concentrations of macronutrients should be expressed as mmol/l
– and
micronutrients as µmol/l
–.
Constituents of Media:
Many elements are needed for plant nutrition and their physiological
functions. Thus, these elements have to be supplied in the culture medium to
support adequate growth of cultures in vitro. A selected list of the elements
and their functions in plants is given in Table 43.2.

The culture media usually contain the following constituents:
1. Inorganic nutrients
2. Carbon and energy sources
3. Organic supplements
4. Growth regulators
5. Solidifying agents
6. pH of medium

Inorganic Nutrients:
The inorganic nutrients consist of macronutrients (concentration >0.5
mmol/l
–) and micronutrients (concentration <0.5 mmol/l
–). A wide range of
mineral salts (elements) supply the macro- and micronutrients. The inorganic
salts in water undergo dissociation and ionization. Consequently, one type of
ion may be contributed by more than one salt. For instance, in MS medium,
K
+ ions are contributed by KNO3 and KH2PO4 while NO3
– ions come from
KNO3 and NH4NO3.
Macronutrient elements:
The six elements namely nitrogen, phosphorus, potassium, calcium,
magnesium and sulfur are the essential macronutrients for tissue culture. The
ideal concentration of nitrogen and potassium is around 25 mmol I
-1 while for
calcium, phosphorus, sulfur and magnesium, it is in the range of 1-3 mmol I
–.
For the supply of nitrogen in the medium, nitrates and ammonium salts are
together used.
Micronutrients:
Although their requirement is in minute quantities, micronutrients are
essential for plant cells and tissues. These include iron, manganese, zinc,
boron, copper and molybdenum. Among the microelements, iron requirement
is very critical. Chelated forms of iron and copper are commonly used in
culture media.
Carbon and Energy Sources:
Plant cells and tissues in the culture medium are heterotrophic and therefore,
are dependent on the external carbon for energy. Among the energy sources,
sucrose is the most preferred. During the course of sterilization (by
autoclaving) of the medium, sucrose gets hydrolysed to glucose and fructose.
The plant cells in culture first utilize glucose and then fructose. In fact, glucose
or fructose can be directly used in the culture media. It may be noted that for
energy supply, glucose is as efficient as sucrose while fructose is less efficient.
It is a common observation that cultures grow better on a medium with
autoclaved sucrose than on a medium with filter-sterilized sucrose. This clearly
indicates that the hydrolysed products of sucrose (particularly glucose) are
efficient sources of energy. Direct use of fructose in the medium subjected to
autoclaving, is found to be detrimental to the growth of plant cells. Besides
sucrose and glucose, other carbohydrates such as lactose, maltose, galactose,
raffinose, trehalose and cellobiose have been used in culture media but with a
very limited success.

Organic Supplements:
The organic supplements include vitamins, amino acids, organic acids, organic
extracts, activated charcoal and antibiotics.
Vitamins:
Plant cells and tissues in culture (like the natural plants) are capable of
synthesizing vitamins but in suboptimal quantities, inadequate to support
growth. Therefore the medium should be supplemented with vitamins to
achieve good growth of cells. The vitamins added to the media include
thiamine, riboflavin, niacin, pyridoxine, folic acid, pantothenic acid, biotin,
ascorbic acid, myoinositol, Para amino benzoic acid and vitamin E.
Amino acids:
Although the cultured plant cells can synthesize amino acids to a certain
extent, media supplemented with amino acids stimulate cell growth and help
in establishment of cells lines. Further, organic nitrogen (in the form of amino
acids such as L-glutamine, L-asparagine, L- arginine, L-cysteine) is more
readily taken up than inorganic nitrogen by the plant cells.
Organic acids:
Addition of Krebs cycle intermediates such as citrate, malate, succinate or
fumarate allow the growth of plant cells. Pyruvate also enhances the growth of
cultured cells.
Organic extracts:
It has been a practice to supplement culture media with organic extracts such
as yeast, casein hydrolysate, coconut milk, orange juice, tomato juice and
potato extract. It is however, preferable to avoid the use of natural extracts due
to high variations in the quality and quantity of growth promoting factors in
them. In recent years, natural extracts have been replaced by specific organic
compounds e.g., replacement of yeast extract by L-asparagine; replacement of
fruit extracts by L-glutamine.
Activated charcoal:
Supplementation of the medium with activated charcoal stimulates the growth
and differentiation of certain plant cells (carrot, tomato, orchids). Some
toxic/inhibitory compounds (e.g. phenols) produced by cultured plants are
removed (by adsorption) by activated charcoal, and this facilitates efficient cell
growth in cultures. Addition of activated charcoal to certain cultures (tobacco,
soybean) is found to be inhibitory, probably due to adsorption of growth
stimulants such as phytohormones.

Antibiotics:
It is sometimes necessary to add antibiotics to the medium to prevent the
growth of microorganisms. For this purpose, low concentrations of
streptomycin or kanamycin are used. As far as possible, addition of antibiotics
to the medium is avoided as they have an inhibitory influence on the cell
growth.
Growth Regulators:
Plant hormones or phytohormones are a group of natural organic compounds
that promote growth, development and differentiation of plants. Four broad
classes of growth regulators or hormones are used for culture of plant cells-
auxins, cytokinins, gibberellins (Fig. 43.1) and abscisic acid. They promote
growth, differentiation and organogenesis of plant tissues in cultures.

Auxins:
Auxins induce cell division, cell elongation, and formation of callus in cultures.
At a low concentration, auxins promote root formation while at a high
concentration callus formation occurs. A selected list of auxins used in tissue
cultures is given in Table 43.3.

Among the auxins, 2, 4-dichlorophenoxy acetic acid is most effective and is
widely used in culture media.
Cytokinins:
Chemically, cytokinins are derivatives of a purine namely adenine. These
adenine derivatives are involved in cell division, shoot differentiation and
somatic embryo formation. Cytokinins promote RNA synthesis and thus
stimulate protein and enzyme activities in tissues. The most commonly used
cytokinins are given in Table 43.3. Among the cytokinins, kinetin and benzyl-
amino purine are frequently used in culture media.
Ratio of auxins and cytokinins:
The relative concentrations of the growth factors namely auxins and cytokinins
are crucial for the morphogenesis of culture systems. When the ratio of auxins
to cytokinins is high, embryogenesis, callus initiation and root initiation occur.
On the other hand, for axillary and shoot proliferation, the ratio of auxins to
cytokinins is low. For all practical purposes, it is considered that the formation
and maintenance of callus cultures require both auxin and cytokinin, while
auxin is needed for root culture and cytokinin for shoot culture. The actual

concentrations of the growth regulators in culture media are variable
depending on the type of tissue explant and the plant species.
Gibberellins:
About 20 different gibberellins have been identified as growth regulators. Of
these, gibberellin A3 (GA3) is the most commonly used for tissue culture.
GA3 promotes growth of cultured cells, enhances callus growth and induces
dwarf plantlets to elongate. Gibberellins are capable of promoting or inhibiting
tissue cultures, depending on the plant species. They usually inhibit
adventitious root and shoot formation.
Abscisic acid (ABA):
The callus growth of cultures may be stimulated or inhibited by ABA. This
largely depends on the nature of the plant species. Abscisic acid is an
important growth regulation for induction of embryogenesis.
Solidifying Agents:
For the preparation of semisolid or solid tissue culture media, solidifying or
gelling agents are required. In fact, solidifying agents extend support to tissues
growing in the static conditions.
Agar:
Agar, a polysaccharide obtained from seaweeds, is most commonly
used as a gelling agent for the following reasons.
1. It does not react with media constituents.
2. It is not digested by plant enzymes and is stable at culture temperature.
Agar at a concentration of 0.5 to 1% in the medium can form a gel.
Gelatin:
It is used at a high concentration (10%) with a limited success. This is mainly
because gelatin melts at low temperature (25°C), and consequently the gelling
property is lost.
Other gelling agents:
Bio-gel (polyacrylamide pellets), phytagel, gelrite and purified agarose are
other solidifying agents, although less frequently used. It is in fact
advantageous to use synthetic gelling compounds, since they can form gels at a
relatively low concentration (1.0 to 2.5 g l
-1).
pH of medium:
The optimal pH for most tissue cultures is in the range of 5.0-6.0. The pH
generally falls by 0.3-0.5 units after autoclaving. Before sterilization, pH can be

adjusted to the required optimal level while preparing the medium. It is
usually not necessary to use buffers for the pH maintenance of culture media.
At a pH higher than 7.0 and lower than 4.5, the plant cells stop growing in
cultures. If the pH falls during the plant tissue culture, then fresh medium
should be prepared. In general, pH above 6.0 gives the medium hard
appearance, while pH below 5.0 does not allow gelling of the medium.
Preparation of Media:
The general methodology for a medium preparation involves preparation of
stock solutions (in the range of 10x to 10Ox concentrations) using high purity
chemicals and demineralized water. The stock solutions can be stored (in glass
or plastic containers) frozen and used as and when required. Most of the
growth regulators are not soluble in water. They have to be dissolved in NaOH
or alcohol.
Dry powders in Media Preparation:
The conventional procedure for media preparation is tedious and time
consuming. Now a days, plant tissue culture media are commercially prepared,
and are available in the market as dry powders. The requisite medium can be
prepared by dissolving the powder in a glass distilled or demineralized water.
Sugar, organic supplements and agar (melted) are added, pH adjusted and the
medium diluted to a final volume (usually 1 litre).
Sterilization of Media:
The culture medium is usually sterilized in an autoclave at 121°C and 15 psi for
20 minutes. Hormones and other heat sensitive organic compounds are filter-
sterilized, and added to the autoclaved medium.
Selection of a Suitable Medium:
In order to select a suitable medium for a particular plant culture system, it is
customary to start with a known medium (e.g. MS medium, B5 medium) and
then develop a new medium with the desired characteristics. Among the
constituents of a medium, growth regulators (auxins, cytokinins) are highly
variable depending on the culture system.
In practice, 3-5 different concentrations of growth regulators in different
combinations are used and the best among them are selected. For the selection
of appropriate concentrations of minerals and organic constituents in the
medium, similar approach referred above, can be employed.

Medium-utmost Important for Culture:
For tissue culture techniques, it is absolutely essential that the medium
preparation and composition are carefully followed. Any mistake in the
preparation of the medium is likely to do a great harm to the culture system as
a whole.
Plant hormones and growth regulat ors are
chemicals that affect:
1 Flowering.
2 Aging.
3 Root growth.
4 Distortion and killing of organs.
5 Prevention or promotion of stem elongation.
6 Color enhancement of fruit.
7 Prevention of leafing, leaf fall or both.
8 Many other conditions.
Very small concentrations of these substances produce major
growth changes.
Compound Effect/Use
Gibberellic acid (GA)
Stimulates cell division and elongation,
breaks dormancy, speeds germination
Ethylene gas (CH2)
Ripening agent; stimulates leaf and fruit
abscission
Indoleacetic acid (IAA)
Stimulates apical dominance, rooting,
and leaf abscission
Indolebutyric acid
(IBA)
Stimulates root growth
Naphthalene acetic
acid (NAA)
Stimulates root growth, slows
respiration (used as a dip on holly)
Growth retardants
(Alar, B-9, Cycocel,
Prevent stem elongation in selected
crops (e.g., chrysanthemums,

Arest) poinsettias, and lilies)
Herbicides (2,4-D,
etc.)
Distorts plant growth; selective and
nonselective materials used for killing
unwanted plants
Hormones are produced naturally by plants, while plant
growth regulators are applied to plants by humans. Plant
growth regulators may be synthetic compounds, such as IBA
and Cycocel, that mimic naturally occurring plant hormones, or
they may be natural hormones that were extracted from plant
tissue, such as IAA.
Applied concentrations of these substances usually are
measured in parts per million (ppm) and in some cases parts
per billion (ppb). These growth-regulating substances most
often are applied as a spray to foliage or as a liquid drench to
the soil around a plant's base. Generally, their effects are short-
lived, and they may need to be reapplied in order to achieve the
desired effect.
There are five groups of plant-growth-regulating compounds:
auxin, gibberellin (GA), cytokinin, ethylene, and abscisic acid
(ABA). For the most part, each group contains both naturally
occurring hormones and synthetic substances.
AUXIN:

causes several responses in plants:

1. Bending toward a light source (phototropism).
2. Downward root growth in response to gravity (geotropism).
3. Promotion of apical dominance (the tendency of an apical
bud to produce hormones that suppress the growth of the buds
below it on the stem).
4. Flower formation.
5. Fruit set and growth.
6. Formation of adventitious roots.

Auxin is the active ingredient in most rooting compounds in
which cuttings are dipped during vegetative propagation.
GIBBERLIN:

stimulate cell division and elongation, break seed dormancy,
and speed germination. The seeds of some species are difficult
to germinate; you can soak them in a GA solution to get them
started.

CYTOKININS:
Unlike other hormones, cytokinins are found in both plants and
animals. They stimulate cell division and often are included in
the sterile media used for growing plants from tissue culture. If
a medium's mix of growth-regulating compounds is high in
cytokinins and low in auxin, the tissue culture explant (small
plant part) will produce numerous shoots. On the other hand, if
the mix has a high ratio of auxin to cytokinin, the explant will
produce more roots. Cytokinins also are used to delay aging and
death (senescence).

ETHYLENE:

Ethylene is unique in that it is found only in the gaseous form.
It induces ripening, causes leaves to droop (epinasty) and drop
(abscission), and promotes senescence. Plants often increase
ethylene production in response to stress, and ethylene often is
found in high concentrations within cells at the end of a plant's
life. The increased ethylene in leaf tissue in the fall is part of the
reason leaves fall off trees. Ethylene also is used to ripen fruit
(e.g., green bananas).

ABSCISIC ACID:

Abscisic acid (ABA) is a general plant-growth inhibitor. It
induces dormancy and prevents seeds from germinating; causes
abscission of leaves, fruits, and flowers; and causes stomata to

close. High concentrations of ABA in guard cells during periods
of drought stress probably play a role in stomatal closure.

STERILIZATION TECHNIQUES:
A) Dry Heat Sterilization – Principle and Uses
What is dry heat sterilization? It is the process of killing bacterial
spores and microorganisms using a high temperature.
This type of sterilization method is used on items that cannot get
wet such as powders, oils, and the likes.
Commonly used instruments for dry heat sterilization are the
following:
1.Hot air oven
2. Microwave
3. Radiation
4. Flaming
5. Incineration/burning
6. Glass bead sterilizer
7. Bunsen burner

Dry heat sterilization has many types such as the following:
Hot air oven
It is a common form of dry heat sterilization used in the workplace. It is
basically an oven that can hold several objects. It is closed for a specific
timeframe to sterilize the objects inside it. The desired temperature
should be achieved to fully maximize the sterilization process.
Hot air oven sterilizes the object inside it without causing harm to the
objects and to the environment. (1, 2, and 3)

The principle of hot air oven dry heat sterilization
Sterilization is achieved by means of conduction. The heat in the oven is
absorbed by the item inside it and passes towards the center of the item
layer by layer. For the item to be fully sterilized, it needs to reach the
required temperature.
What dry heat sterilization does is it inflicts damage by oxidizing
molecules leading to the organism’s death. Resistant spores are killed by
exposing them at a higher temperature for a long period of time.

There are two types of hot air oven. These are the following:
1. Static air hot air oven – The oven is heated using the coils on the
bottom. It would take some time for the oven to achieve its desired
temperature. There is also a possibility that the desired heat will not be
eventually distributed throughout the oven.

2. Forced air hot air oven – it has a motorized blower that evenly
distributes the heat throughout the oven. Another advantage of this hot air
oven is that it does not take long to heat the entire oven.

What are the advantages of dry heat sterilization?
It is a non-toxic way of sterilizing things.
It is safe for the environment.
It gently and thoroughly penetrates materials.
It is compatible with metal and sharp objects because it is non-corrosive.
It has a low operating cost. (4, 5)

What are the disadvantages of dry heat sterilization?
Such method of sterilization takes time because of the slow rate of heat
penetration.
It requires an extremely high temperature to thoroughly kill resistant
microbes, which makes it not suitable for rubber and plastic materials.
Overexposure to heat may ruin the items being sterilized for. (5, 6)

Things commonly sterilized using dry heat sterilization method
Metal instruments
Glassware
Syringes
Paper-wrapped items
Powder
Fats
Anhydrous oils (7)

Dry heat sterilization features
1. It does not require human intervention. What the human needs to
do is to simply set the required temperature and time. As a matter of
fact, some dry heat sterilization equipment only requires you to
choose a particular setting and after which the machine will do all
the necessary work.

2. .Dry heat sterilizers come with a special ventilation system. The air
in the chamber is heated evenly thereby preserving sterilized
objects. The temperature inside the sterilizer is supported and the
possibility of overheating is eliminated.
3. It comes with a reserve system dependent on the thermostat and
works independently from the primary system.
4. You can sterilize both packed and unpacked objects. (3, 5, 7, and 8)

Difference between dry heat sterilization and moist heat sterilization
Both dry heat and moist heat are used to sterilize objects. However, there
is a huge difference between the two. Dry heat uses dry air of high
temperature while moist heat sterilization uses a low temperature and a
high pressure of water. Dry heat sterilization takes more time than the
heat moist sterilization.
The two comes with advantages and disadvantages. Moist heat
sterilization requires low temperature and less time to complete. It is also
low cost and non-toxic. However, it cannot sterilize heat-sensitive
instruments.

STERILIZATION TECHNIQUES:

Since it is using water, the instruments or items being sterilized remain
wet which increases the possibility of rusting. Repeated exposure to
moisture may damage the items being sterilized. The advantages and
disadvantages of dry heat sterilization are already mentioned above.

WET HEAT (Autoclaving)
The method of choice for sterilisation in most labs is autoclaving; using pressurised
steam to heat the material to be sterilised. This is a very effective method that kills all
microbes, spores and viruses, although for some specific bugs, especially high
temperatures or incubation times are required.
Autoclaving kills microbes by hydrolysis and coagulation of cellular proteins, which is
efficiently achieved by intense heat in the presence of water.
The intense heat comes from the steam. Pressurised steam has a high latent heat; at
100degC it holds 7 times more heat than water at the same temperature. This heat is
liberated upon contact with the cooler surface of the material to be sterilised, allowing
rapid delivery of heat and good penetration of dense materials.
At these temperatures, water does a great job of hydrolysing proteins… so those bugs
don’t stand a chance.
DRY HEAT (Flaming, baking)
Dry heating has one crucial difference from autoclaving. You’ve guessed it – there’s no
water, so protein hydrolysis can’t take place.
Instead, dry heat tends to kill microbes by oxidation of cellular components. This
requires more energy than protein hydrolysis so higher temperatures are required for
efficient sterilization by dry heat.
For example sterilisation can normally be achieved in 15 minutes by autoclaving at
121degC, whereas dry heating would generally need a temperature of 160degC to
sterilize in a similar amount of time.
FILTRATION
Filtration is a great way of quickly sterilizing solutions without heating. Filters, of course,
work by passing the solution through a filter with a pore diameter that is too small for
microbes to pass through.
Filters can be scintered glass funnels made from heat-fused glass particles or, more
commonly these days, membrane filters made from cellulose esters. For removal of
bacteria, filters with an average pore diameter of 0.2um is normally used.
But remember, viruses and phage can pass through these filters so filtration is not a
good option if these are a concern.
SOLVENTS

Ethanol is commonly used as a disinfectant, although since isopropanol is a better
solvent for fat it is probably a better option.
Both work by denaturing proteins through a process that requires water, so they must
be diluted to 60-90% in water to be effective.
Again, a it’s important to remember that although ethanol and IPA are good at killing
microbial cells, they have no effect on spores.
RADIATION
UV, x-rays and gamma rays are all types of electromagnetic radiation that have
profoundly damaging effects on DNA, so make excellent tools for sterilization.
The main difference between them, in terms of their effectiveness, is their penetration.
UV has limited penetration in air so sterilisation only occurs in a fairly small area around
the lamp. However, it is relatively safe and is quite useful for sterilising small areas, like
laminar flow hoods.
X-rays and gamma rays are far more penetrating, which makes them more dangerous
but very effective for large scale cold sterilization of plastic items (e.g. syringes) during
manufacturing.
So those are some of the main methods for sterilization I can think of. If I’ve missed
any, please feel free to let me know in the comments section.
Sterilisation Instruments:
Routinely Used Instruments for Sterilisation:
a. Physical Methods – Dry Heat:
i. Hot air oven:
The oven is heated by electricity and has a thermostat to maintain the air
temperature constant (160°C for 1 hour). It is the best method to sterilize dry
glass water, cotton wool plugged test tubes, Petri dishes, flasks, pipettes,
swabs, powder, fat or grease). All glassware’s should be dry, and wrapped in
Kraft paper to soak any water drop on the glassware.
The oven should be loaded in such a way that the air can circulate through the
load. The temperature should be raised slowly in the course of 1-2 hours to
reach 160°C—which must be maintained for 1 hour. This is the holding period.
Finally, the oven is allowed to cool gradually for 2 hours before the door is
opened, otherwise the glassware’s may crack due to sudden atmospheric
cooling.
ii. Infrared radiation:
The infrared rays are directed on the object to be sterilised at a temperature of
180°C as a means of sterilising surgical instruments.

b. Moist Heat:
Below 100°C:
i. Constant temperature water bath:
It is a metal box containing a heating element and a thermostat that keeps the
water in the box at a constant temperature (56°C or 60°C). During the
preparation of bacterial vaccine, the bacteria in suspension are killed in the
water bath (vaccine bath) at a temperature of 60°C for 1 hour.
The sterilisation of serum or body fluids containing coagulable proteins is
effected at 56°C for 1 hour in water bath. The temperature above 59°C may
cause inspissation (coagulation).

2. Pasteurisation of milk can be done by two methods:
(a) Holder method (63°C for 30 minutes) or
(b) Flash method (72° for 20 seconds) in which tubercle bacilli,
Brucella abortus and Salmonella are destroyed.
3. Inspissator:
It consists of a water jacketed copper box. The temperature is between 75°-
85°C in which the protein is completely solidified.
The serum (Loeffler serum) and egg (Dorset egg or Lowenstein Jensen
medium) in screw-capped (MacCartney) bottles are placed in special racks so
that the tubes are in slanting position to form slopes; water vapour can enter
the interior of the inspissator, the medium is kept moist, sterilised at 75°C for
45 minutes intermittently for three days and solidified.

Exposure at 100°C for 20-45 minutes on each of the three successive days,
sometimes referred to as Fractional sterilisation (Tyndallisation), is employed
to sterilize sugar media, gelatin media etc.
Above 100°C:
Boiling water in fish kettle (100°C for 10 minutes) is sufficient to kill the non-
spore forming bacilli. It does not ensure sterility and is used for rubber
stoppers, instruments (scalpels, forceps, scissors), metal and glass type
syringes.
Steam at 100°C is used for the sterilisation of culture media (nutrient broth
and agar), but it is not as effective as autoclaving. Koch or Arnold steamer
(sterilizer) heated by gas or electricity is employed. It is a vertical cylinder with
a conical lid having a small opening for escaping steam, i.e. steam without
pressure.
Above 100°C:
Principle:
Water boils when its vapour pressure equals the pressure of the surrounding
atmosphere. This occurs at 100°C for normal atmospheric pressure (i.e. 760
mm Hg = 14.7 lb. per square inch absolute pressure).
Thus, when vapour boils within a closed vessel at increased pressure, the
temperature at which it boils will rise above 100°C.
This principle is employed in “Autoclave” which provides heat at
temperatures above 100°C. Autoclaving is the reliable method most widely
used for sterilisation of culture media and surgical supplies.
All parts of the load to be sterilised should be permeated by the
steam.
1. The simple form of laboratory autoclave, the so-called “pressure cooker
type” (Fig. 4.1), consists of a vertical cylinder of gun metal or stainless steel.
Its size is 18 inch (45 cm) in diameter and 30 inch (75 cm), in length. The lid
(or door) is fastened by screw clamps and is rendered airtight by means of
asbestos washer.
The cylinder contains water up to a certain level and is heated electrically
below the cylinder. The bottles, tubes, culture media etc. to be sterilised are
placed on a perforated tray situated above the water level. This apparatus is
furnished on its lid with a discharge tap for air and steam, a pressure gauge,
and a safety valve.

Direction for using the simple autoclave:
Water should be sufficient in the cylinder. Insert the material to be sterilised
and turn on the heater. Place the lid in position, open the discharge tap and
screw down the lid. Adjust the safety valve to the required pressure. Allow the
steam and air mixture to escape freely until all the air has been eliminated
from the autoclave.
Now close the discharge tap. The steam pressure up to 15 lb. pressure per sq.
inch for 121°C should be maintained for 15 minutes. This is the holding period.
Then turn off the heater and allow the autoclave to cool until the pressure is 0
lb. per sq. inch. To avoid a violent explosion, do not open the autoclave when
its inside pressure is still high.

2. Steam jacketed autoclave (hospital type):
This autoclave is horizontal and is made of rust-less metal. At its front is a
swing door fastened by a “capstan head” which operates radially and
remains locked. A pressure locked safety door is a valuable guard against a
dangerous explosion through premature opening by the operator.
The hospital type autoclave (Fig. 4.2) has:
(a) A supply of steam from external source;
(b) Steam jacket;
(c) A channel to discharge air and steam;
(d) Steam trap with thermometer to control the discharge;
(e) A vacuum system to assist the drying of the load;
(f) A cooling system to hasten the cooling.

To test the efficiency of autoclaving:
Two methods (chemical and spore indicators) are available to detect the
efficiency of autoclaving.
(i) Chemical indicators:
It may be placed inside the load. Browne’s sterilizer control tubes contain a red
solution that turns green when heated at 115°C for 25 minutes (type 1) or 15
minutes (type 2) or at 160°C for 60 minutes (type 3). Bowie Dick tape, applied
to packs and articles in the autoclave, develops diagonal lines when exposed to
the sterilising temperature for the correct time.
(ii) Spore indicators:
A preparation of dried spores of Bacillus stearothermophilus is placed within
the load in the autoclave and after autoclaving it is tested for viability on
transfer to culture media; its spores are killed at 121°C in about 12 minutes.
c. Radiation:
Non- Ionising Radiation
Ultraviolet rays from suitably shielded lamps have been used to reduce the
number of bacteria in the atmosphere. Infrared radiation is used for rapid
mass sterilisation of glass syringes. Ionising radiation includes high speed
electrons; X-rays and gamma rays (short X-rays).

In sufficient dose, these radiations are lethal to all cells including bacteria by
damaging DNA. Spores are generally resistant. It is employed commercially for
sterilisation of plastic syringes, rubber and catheters that are unable to
withstand the heat. It is too expensive for the hospital use. This method is
called cold sterilisation. Plastic syringes can also be sterilised by ethylene oxide
gas.
d. Filtration:
It is possible to render the fluids, bacterial cultures, sugar by passing through
the special filters with pore size of less than 0.75 pm. In general, sterilising
filters render liquid free from bacteria, but not from mycoplasma or virus.
Thus, the serum “sterilised” by Seitz filtration must not be regarded as safe
for clinical use.
Types of filter used in bacteriological work include:
(a) Earthenware candles (Berkefeld Chamberland);
(b) Asbestos disks (Seitz filter);
(c) Sintered glass filters;
(d) In recent years, cellulose membrane filters are used to separate viruses of
different sizes.
Seitz filter:
It consists of a disk of an asbestos through which the fluid is filtered. The disk
is inserted into the metal holder fitted to a filtering flask. Before use, the whole
apparatus should be sterilised by autoclave (Fig. 4.3).

The development of high efficiency particulate air (HEPA) filters or fiberglass
filters has made it possible to deliver clean air to an enclosure (cubicle or
room). This type of air filtration together with a system of Laminar Airflow is
now recommended as air disinfection and used very extensively to produce
dust and bacteria-free air in vaccine sterility testing laboratory; but it is
expensive.
e. Chemical Methods:
Common chemical disinfectants used:

 HOL 
 U…L 

plant tissue culture- all notes

About This Presentation

Slide Content

Tags

Categories

Download

Quick Actions

Statistics

Related Slideshows

plant tissue culture- all notes

About This Presentation

Slide Content

Slide 2

Slide 3

Slide 4

Slide 5

Slide 6

Slide 7

Slide 8

Slide 9

Slide 10

Slide 11

Slide 12

Slide 13

Slide 14

Slide 15

Slide 16

Slide 17

Slide 18

Slide 19

Slide 20

Slide 21

Slide 22

Slide 23

Slide 24

Slide 25

Slide 26

Slide 27

Slide 28

Slide 29

Slide 30

Slide 31

Slide 32

Slide 33

Slide 34

Slide 35

Slide 36

Slide 37

Slide 38

Slide 39

Slide 40

Slide 41

Slide 42

Slide 43

Slide 44

Slide 45

Slide 46

Slide 47

Slide 48

Slide 49

Slide 50

Slide 51

Slide 52

Slide 53

Slide 54

Slide 55

Slide 56

Slide 57

Slide 58

Slide 59

Slide 60

Slide 61

Slide 62

Slide 63

Slide 64

Slide 65

Tags

Categories

Download

Quick Actions

Statistics

Related Slideshows

Pray For The Peace Of Jerusalem and You Will Prosper

Don_t_Waste_Your_Life_God.....powerpoint

VILLASUR_FACTORS_TO_CONSIDER_IN_PLATING_SALAD_10-13.pdf

Fertility awareness methods for women in the society

Chapter 5 Arithmetic Functions Computer Organisation and Architecture

syakira bhasa inggris (1) (1).pptx.......