To achieve the target of creating a new plant or a plant with desired characteristics, tissue culture is often coupled with recombinant DNA technology. The techniques of plant tissue culture have largely helped in the green revolution by improving the crop yield and quality. The knowledge obtained from plant tissue cultures has contributed to our understanding of metabolism, growth, differentiation and morphogenesis of plant cells. Further, developments in tissue culture have helped to produce several pathogen-free plants, besides the synthesis of many biologically important compounds, including pharmaceuticals. Because of the wide range of applications, plant tissue culture attracts the attention of molecular biologists, plant breeders and industrialists.

1. Plant Tissue Culture
Dr. Naveen Gaurav
Associate Professor and Head
Department of Biotechnology
Shri Guru Ram Rai University
Dehradun

2. Plant Tissue Culture:
Plant tissue culture broadly refers to the in vitro cultivation of plants, seeds and various
parts of the plants (organs, embryos, tissues, single cells, protoplasts).
The cultivation process is invariably carried out in a nutrient culture medium under aseptic
conditions. Plant cells have certain advantages over animal cells in culture systems. Unlike
animal cells, highly mature and differentiated plant cells retain the ability of totipotency i.e.
the ability of change to meristematic state and differentiate into a whole plant.
Benefits of Plant Tissue Culture:
Plant tissue culture is one of the most rapidly growing areas of biotechnology because of its
high potential to develop improved crops and ornamental plants. With the advances made
in the tissue culture technology, it is now possible to regenerate species of any plant in the
laboratory.
To achieve the target of creating a new plant or a plant with desired characteristics, tissue
culture is often coupled with recombinant DNA technology. The techniques of plant tissue
culture have largely helped in the green revolution by improving the crop yield and quality.
The knowledge obtained from plant tissue cultures has contributed to our understanding of
metabolism, growth, differentiation and morphogenesis of plant cells. Further,
developments in tissue culture have helped to produce several pathogen-free plants,
besides the synthesis of many biologically important compounds, including
pharmaceuticals. Because of the wide range of applications, plant tissue culture attracts
the attention of molecular biologists, plant breeders and industrialists.

3. Basic Structure and Growth of a Plant:
An adult plant basically consists of a stem and a root, each with many branches (Fig. 42.1).
Both the stem and root are characterized by the presence of apical growth regions which
are composed of meristematic cells. These cells are the primary source for all the cell
types of a plant.
The plant growth and development occur in two different ways:
1. Determinate growth:
This is characterized by ceasation of growth as the plant parts attain certain size and
shape, e.g., leaves, flowers, fruits.
2. Indeterminate growth:
This refers to the continuous growth of roots and stems under suitable conditions. It is
possible due to the presence of meristems (in stems and roots) which can proliferate
continuously. As the seed germinates and seedling emerges, the meristematic cells of the
root apex multiply. Above the root apex, the cells grow in length without multiplication.
Some of the elongated cells of the outer layer develop into root hairs to absorb water and
nutrients from the soil. As the plant grows, root cells differentiate into phloem and xylem.
Phloem is responsible for the absorption of nutrients while xylem absorbs water.
The meristematic cells of the shoot apex divide leading to the growth of stem. Some of the
stem cells differentiate and develop into leaf primordia, and then leaves. Axillary buds
present between the leaf primordia and elongated stem also possess meristems which can
multiply and give rise to branches and flowers.
A diagrammatic view of a plant and a flower are respectively depicted in Fig. 42.1 and Fig.
42.2.

5. Conventional Plant Breeding and Plant Tissue Culture:
Since the time immemorial, man has been closely involved in the improvement of plants to
meet his basic needs. The conventional methods employed for crop improvement are very
tedious and longtime processes (sometimes decades). Further, in the conventional
breeding methods, it is not possible to introduce desired genes to generate new characters
or products.
With the developments in plant tissue culture, it is now possible to reduce the time for the
creation of new plants with desired characteristics, transfer of new genes into plant cells
and large scale production of commercially important products.
Terms Used in Tissue Culture:
A selected list of the most commonly used terms in tissue culture are briefly explained
Explant:
An excised piece of differentiated tissue or organ is regarded as an explant. The explant
may be taken from any part of the plant body e.g., leaf, stem, root.
Callus:The unorganized and undifferentiated mass of plant cells is referred to as callus.
Generally, when plant cells are cultured in a suitable medium, they divide to form callus
i.e., a mass of parenchymatous cells.
Dedifferentiation:
The phenomenon of mature cells reverting to meristematic state to produce callus is
dedifferentiation. Dedifferentiation is possible since the non- dividing quiescent cells of the
explant, when grown in a suitable culture medium revert to meristematic state.
Re-differentiation:
The ability of the callus cells to differentiate into a plant organ or a whole plant is regarded
as re-differentiation.

6. Totipotency:
The ability of an individual cell to develop into a whole plant is referred to as cellular
totipotency. The inherent characteristic features of plant cells namely dedifferentiation and
re-differentiation are responsible for the phenomenon of totipotency. The other terms used
in plant tissue culture are explained at appropriate places.
Brief History of Plant Tissue Culture:
About 250 years ago (1756), Henri-Louis Duhamel du Monceau demonstrated callus
formation on the decorticated regions of elm plants. Many botanists regard this work as the
forward for the discovery of plant tissue culture. In 1853, Trecul published pictures of callus
formation in plants.
German botanist Gottlieb Haberlandt (1902), regarded as the father of plant tissue culture,
first developed the concept of in vitro cell culture. He was the first to culture isolated and
fully differentiated plant cells in a nutrient medium. During 1934-1940, three scientists
namely Gautheret, White and Nobecourt largely contributed to the developments made in
plant tissue culture.
Good progress and rapid developments occurred after 1940 in plant tissue culture
techniques. Steward and Reinert (1959) first discovered somatic embryo production in vitro.
Maheswari and Guha (1964) from India were the first to develop anther culture and poller
culture for the production of haploid plants.
Types of Culture:
There are different types of plant tissue culture techniques, mainly based on the explant
used (Fig. 42.3).

7. Callus culture:
This involves the culture of differentiated tissue from explant which dedifferentiates in vitro to form
callus.
Organ culture:
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Culture of isolated plant organs is referred to as organ culture. The organ used may be embryo, seed,
root, endosperm, anther, ovary, ovule, meristem (shoot tip) or nucellus. The organ culture may be
organized or unorganized.
Organized organ culture:
When a well-organized structure of a plant (seed, embryo) is used in culture, it is referred to as
organized culture. In this type of culture, the characteristic individual organ structure is maintained
and the progeny formed is similar in structure as that of the original organ.
Unorganized organ culture:
This involves the isolation of cells or tissues of a part of the organ, and their culture in vitro.
Unorganized culture results in the formation of callus. The callus can be dispersed into aggregates of
cells and/or single cells to give a suspension culture.
Cell culture:
The culture of isolated individual cells, obtained from an explant tissue or callus is regarded as cell
culture. These cultures are carried out in dispension medium and are referred to as cell suspension
cultures.
Protoplast culture:
Plant protoplasts (i.e., cells devoid of cell walls) are also used in the laboratory for culture.

8. Basic Technique of Plant Tissue Culture:
The general procedure adopted for isolation and culture of plant tissues is depicted in Fig.
42.4

9. The requisite explants (buds, stem, seeds) are trimmed and then subjected to sterilization in a
detergent solution. After washing in sterile distilled water, the explants are placed in a suitable culture
medium (liquid or semisolid form) and incubated. This results in the establishment of culture. The
mother cultures can be subdivided, as frequently as needed, to give daughter cultures.
The most important aspect of in vitro culture technique is to carry out all the operations under aseptic
conditions. Bacteria and fungi are the most common contaminants in plant tissue culture. They grow
much faster in culture and often kill the plant tissue.Further, the contaminants also produce certain
compounds which are toxic to the plant tissue. Therefore, it is absolutely essential that aseptic
conditions are maintained throughout the tissue culture operations. Some of the culture techniques
are described here while a few others are discussed at appropriate places.
Applications of Plant Tissue Cultures: Plant tissue cultures are associated with a wide range of
applications—the most important being the production of pharmaceutical, medicinal and other
industrially important compounds.
In addition, tissue cultures are useful for several other purposes listed below:
1. To study the respiration and metabolism of plants.
2. For the evaluation of organ functions in plants.
3. To study the various plant diseases and work out methods-for their elimination.
4. Single cell clones are useful for genetic, morphological and pathological studies.
5. Embryonic cell suspensions can be used for large scale clonal propagation.
6. Somatic embryos from cell suspensions can be stored for long term in germplasm banks.
7. In the production of variant clones with new characteristics, a phenomenon referred to as soma
clonal variations.
8. Production of haploids (with a single set of chromosomes) for improving crops.
9. Mutant cells can be selected from cultures and used for crop improvement.
10. Immature embryos can be cultured in vitro to produce hybrids, a process referred to as embryo
rescue.

10. 1. Callus Culture:
Callus is the undifferentiated and
unorganized mass of plant cells. It is
basically a tumor tissue which usually
forms on wounds of differentiated
tissues or organs. Callus cells are
parenchymatous in nature although
not truly homogenous. On careful
examination, callus is found to contain
some quantity of differentiated tissue,
besides the bulk of non-differentiated
tissue.
Callus formation in vivo is frequently
observed as a result of wounds at the
cut edges of stems or roots. Invasion
of microorganisms or damage by
insect feeding usually occurs through
callus. An outline of technique used
for callus culture, and initiation of
suspension culture is depicted in Fig.
42.5.

11. Explants for callus culture:
The starting materials (explates) for callus culture may be the differentiated tissue from any
part of the plant (root, stem, leaf, anther, flower etc.). The selected explant tissues may be
at different stages of cell division, cell proliferation and organization into different distinct
specialized structures. If the explant used possesses meristematic cells, then the cell division
and multiplication will be rapid.
Factors Affecting Callus Culture:
Many factors are known to influence callus formation in vitro culture. These include the
source of the explant and its genotype, composition of the medium (MS medium most
commonly used), physical factors (temperature, light etc.) and growth factors. Other
important factors affecting callus culture are — age of the plant, location of explant,
physiology and growth conditions of the plant.
Physical factors:
A temperature in the range of 22-28°C is suitable for adequate callus formation. As regards
the effect of light on callus, it is largely dependent on the plant species-light may be
essential for some plants while darkness is required by others.
Growth regulators:
The growth regulators to the medium strongly influence callus formation. Based on the
nature of the explant and its genotype, and the endogenous content of the hormone, the
requirements of growth regulators may be categorized into 3 groups
1. Auxin alone
2. Cytokinin alone
3. Both auxin and cytokinin.

12. Suspension culture from callus:
Suspension cultures can be initiated by transferring friable callus to liquid nutrient medium (Fig.
42.5). As the medium is liquid in nature, the pieces of callus remain submerged. This creates
anaerobic condition and ultimately the cells may die. For this reason, suspension cultures have to
be agitated by a rotary shaker. Due to agitation, the cells gets dispersed, besides their exposure to
aeration.
Applications of Callus Cultures:
Callus cultures are slow-growth plant culture systems in static medium. This enables to conduct
several studies related to many aspects of plants (growth, differentiation and metabolism) as listed
below.
i. Nutritional requirements of plants.
ii. Cell and organ differentiation.
iii. Development of suspension and protoplast cultures.
iv. Somaclonal variations.
v. Genetic transformations.
vi. Production of secondary metabolites and their regulation.
2. Suspension Cultures — Growth and Subculture:
The isolated cells are grown in suspension cultures. Cell suspensions are maintained by routine
sub-culturing in a fresh medium. For this purpose, the cells are picked up in the early stationary
phase and transferred. As the cells are incubated in suspension cultures, the cells divide and
enlarge.
The incubation period is dependent on:
i. Initial cell density
ii. Duration of lag phase
iii. Growth rate of cells.

13. Among these, cell density is very crucial. The initial cell density used in the subcultures is very
critical, and largely depends on the type of suspension culture being maintained. With low initial
cell densities, the lag phase and log phases of growth get prolonged.
Whenever a new suspension culture is started, it is necessary to determine the optical cell
density in relation the volume of culture medium, so that maximum cell growth can be achieved.
With low cell densities, the culture will not grow well, and requires additional supplementation
of metabolites to the medium. The normal incubation time for the suspension cultures is in the
range of 21-28 days.
3. Types of Suspension Cultures:
There are mainly two types of suspension cultures — batch cultures and continuous cultures.
Batch cultures:
A batch culture is a cell suspension culture grown in a fixed volume of nutrient culture medium.
In batch culture, cell division and cell growth coupled with increase in biomass occur until one of
the factors in the culture environment (nutrient, O2 supply) becomes limiting. The cells exhibit
the following five phases of growth when the cell number in suspension cultures is plotted
against the time of incubation (Fig. 42.6).
1. Lag phase characterized by preparation of cells to divide.
2. Log phase (exponential phase) where the rate of cell multiplication is highest.
3. Linear phase represented by slowness in cell division and increase in cell size expansion.
4. Deceleration phase characterized by decrease in cell division and cell expansion.
5. Stationary phase represented by a constant number of cells and their size.
The batch cultures can be maintained continuously by transferring small amounts of the
suspension medium (with inoculum) to fresh medium at regular intervals (2-3 days). Batch
cultures are characterized by a constant change in the pattern of cell growth and metabolism.
For this reason, these cultures are not ideally suited for the studies related to cellular behaviour.

14. Continuous cultures:
In continuous cultures, there is a regular addition of fresh nutrient medium and draining
out the used medium so that the culture volume is normally constant. These cultures are
carried out in specially designed culture vessels (bioreactors).
Continuous cultures are carried out under defined and controlled conditions—cell density,
nutrients, O2, pH etc. The cells in these cultures are mostly at an exponential phase (log
phase) of growth.
Continuous cultures are of two types—open and closed.
Open continuous cultures:
In these cultures, the inflow of fresh medium is balanced with the outflow of the volume of
spent medium along with the cells. The addition of fresh medium and culture harvest are
so adjusted that the cultures are maintained indefinitely at a constant growth rate. At a
steady state, the rate of cells removed from the cultures equals to the rate of formation of
new cells.
Open continuous culture system is regarded as chemostat if the cellular growth rate and
density are kept constant by limiting a nutrient in the medium (glucose, nitrogen,
phosphorus). In chemostat cultures, except the limiting nutrient, all other nutrients are
kept at higher concentrations. As a result, any increase or decrease in the limiting nutrient
will correspondingly increase or decrease the growth rate of cells.

15. In turbidostat open continuous cultures, addition of fresh medium is done whenever there
is an increase in turbidity so that the suspension culture system is maintained at a fixed
optical density. Thus, in these culture systems, turbidity is preselected on the basis of
biomass density in cultures, and they are maintained by intermittent addition of medium
and washout of cells.
Closed continuous cultures:
In these cultures, the cells are retained while the inflow of fresh medium is balanced with
the outflow of corresponding spent medium. The cells present in the outflowing medium
are separated (mechanically) and added back to the culture system. As a result, there is a
continuous increase in the biomass in closed continuous cultures. These cultures are
useful for studies related to cytodifferentiation, and for the production of certain
secondary metabolites e.g., polysaccharides, coumarins.
4. Synchronization of Suspension Cultures:
In the normal circumstances, the cultured plant cells vary greatly in size, shape, cell cycle
etc., and are said to be asynchronous. Due to variations in the cells, they are not suitable
for genetic, biochemical and physiological studies. For these reasons, synchronization of
cells assumes significance.
Synchronization of cultured cells broadly refers to the organized existence of majority of
cells in the same cell cycle phase simultaneously.
A synchronous culture may be regarded as a culture in which the cell cycles or specific
phase of cycles for majority of cultured cells occurs simultaneously.
Several methods are in use to bring out synchronization of suspension cultures. They may
be broadly divided into physical and chemical methods.

16. Physical methods: The environmental culture growth influencing physical parameters (light,
temperature) and the physical properties of the cell (size) can be carefully monitored to achieve
reasonably good degree of synchronization. A couple of them are described
Cold treatment: When the suspension cultures are subjected to low temperature (around 4°C)
shock synchronization occurs. Cold treatment in combination with nutrient starvation gives better
results.
Selection by volume: The cells in suspension culture can be selected based on the size of the
aggregates, and by this approach, cell synchronization can be achieved.
Chemical methods: The chemical methods for synchronization of suspension cultures include the
use of chemical inhibitors, and deprivation of an essential growth factor (nutrient starvation). By
this approach, the cell cycle can be arrested at a particular stage, and then allowed to occur
simultaneously so that synchronization is achieved.
Chemical inhibition: Inhibitors of DNA synthesis (5-amino uracil, hydroxyurea, 5-
fluorodeoxypurine), when added to the cultures results in the accumulation of cells at G1 phase.
And on removal of the inhibitor, synchronization of cell division occurs.
Colchicine is a strong inhibitor to arrest the growth of cells at metaphase. It inhibits spindle
formation during the metaphase stage of cell division. Exposure to colchicine must be done for a
short period (during the exponential growth phase), as long duration exposure may lead to
mitoses.
tarvation:
When an essential nutrient or growth promoting compound is deprived in suspension cultures,
this results in stationary growth phase. On supplementation of the missing nutrient compound,
cell growth resumption occurs synchronously. Some workers have reported that deprivation and
subsequent addition of growth hormone also induces synchronization of cell cultures.

17. 5. Measurement of Growth of Cultures:
It is necessary to assess the growth of cells in cultures. The parameters selected for the measuring growth of
suspension cultures include cell counting, packed cell volume and weight increase.
Cell counting:
Although cell counting to assess culture growth is reasonably accurate, it is tedious and time consuming. This is because
cells in suspension culture mostly exist as colonies in varying sizes. These cells have to be first disrupted (by treating
with pectinase or chromic acid), separated, and then counted using a haemocytometer.
Packed cell volume:
Packed cell volume (PCV) is expressed as ml of pellet per ml of culture. To determine PCV, a measured volume of
suspension culture is centrifuged (usually at 2000 x g for 5 minutes) and the volume of the pellet or packed cell volume
is recorded. After centrifugation the supernatant can be discarded, the pellet washed, dried overnight and weighed.
This gives cell dry weight.
Cell fresh weight:
The wet cells are collected on a pre-weighed nylon fabric filter (supported in funnel). They are washed to remove the
medium, drained under vacuum and weighed. This gives the fresh weight of cells. However, large samples have to be
used for accurate weights.
6. Measurement of Viability of Cultured Cells:
The viability of cells is the most important factor for the growth of cells. Viability of cultured cells can be measured by
microscopic examination of cells directly or after staining them.
Phase contrast microscopy:
The viable cells can be detected by the presence of healthy nuclei. Phase contrast microscope is used for this purpose.
Evan’s blue staining:
A dilute solution of Evan’s blue (0.025% w/v) dye stains the dead or damaged cells while the living (viable) cells remain
unstained.
Fluorescein diacetate method:
When the cell suspension is incubated with fluorescein diacetate (FDA) at a final concentration of 0.01%, it is cleaved by
esterase enzyme of living cells. As a result, the polar portion of fluorescein which emits green fluorescence under
ultraviolet (UV) light is released. The viable cells can be detected by their fluorescence, since fluorescein accumulates in
the living cells only.

18. Thank you
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