IMPORTANCE AND HISTORICAL VIEW OF PLANT TISSUE CULTURE

Objective
To begin with, one should know the importance of plant tissue culture in the
improvement of useful crop plants and also the ways in which it has helped mankind. Plant
tissue culture forms an integral part of any plant biotechnology activity. It offers an alternative
to conventional vegetative propagation. But, tissue culture requires attention-to-detail and
unless practiced as art and science, the entire process is rather
unforgiving. The various objectives achievable or achieved by plant tissue culture may be
summarized as under:
a. Crop Improvement
As you all understand that for any crop improvement, conventional breeding methods
are employed which involve six to seven generations of selfing and crossing- over to obtain a
pure line. With plant tissue culture techniques, production of haploids through distant crosses
or using pollen, anther or ovary culture, followed by chromosome doubling, reduces this time
to two generations.
b. Micropropagation
Plant tissue culture techniques have also helped in large- scale production of plants
through micropropagation or clonal propagation of plant species. Small amounts of tissue can
be used to raise hundreds or thousands of plants in a continuous process. This is being
utilized by industries in India for commercial production of mainly ornamental plants like
orchids and fruit trees, e.g., banana. Using this method, millions of genetically identical plants
can be obtained from a single bud. This method has, therefore, become an alternative to
vegetative propagation. Shoot tip propagation is exploited intensively in horticulture and the
nurseries for rapid clonal propagation of many dicots, monocots and gymnosperms.
c. Genetic Transformation
Tissue culture, in combination with genetic engineering is very useful in gene transfers.
For example, the transfer of a useful bacterial gene say, cry (crystal protein) gene from Bacillus
thuringiensis, into a plant cell and, ultimately, regeneration of whole plants containing and
expressing this gene (transgenic plants) can be achieved.
d. Production of Pathogen-free Plants
Eradication of virus has been an outstanding contribution of tissue culture technology.
It was found that even in infected plants the cells of shoot tips are either free of virus or carry a
negligible amount of the pathogen. Such shoot tips are cultured
in a suitable culture medium to obtain virus- free plants. This technique is economical and
used very frequently in horticulture, production of virus- free ornamentals etc.

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e. Production of Secondary Metabolites
Cultured plant cells are also known to produce biochemicals [secondary metabolites]
like, alkaloids, terpenoids, phenyl propanoids etc. of interest. The technology is now available
to the industry. The commercial production of ‘shikonin’[a naphthoquinone] from cell cultures
of Lithospermum erythrorhizon, has been particularly encouraging.
What does the Term Tissue Culture Mean?
The term‘Tissue Culture’ is commonly used in a very wide sense to include in vitro
aseptic culture of plant cells, tissues and organs. The in vitro cultivation of plant cells
produces an unorganized mass of cells called ‘Callus’ which, depending upon the medium of
culture gives rise to roots, somatic embryos, shoots, etc. Another form of cell culture is, in vitro
culture of single or relatively small groups of plant cells in a liquid medium. Such cultures are
known as ‘Suspension’ Cultures’. Sometimes, organized structures like root tips, shoot tips,
embryos etc. are cultured in vitro to obtain their development as organized structures. These
are called ‘Organ Cultures’.
Development of the science of tissue culture is historically linked to the cell and
subsequent propounding of the ‘cell theory’. The in vitro techniques were developed initially to
demonstrate the totipotency of plant cells predicted by Haberlandt in 1902. Totipotency is the
ability of a plant cell to develop into a complete plant. In 1902, Haberlandt reported culture of
isolated single palisade cells from leaves in Knop’s salt solution enriched with sucrose. Cells
were able to synthesise starch as well as increase in size and survived for several weeks, but
failed to divide. He realized that ‘asepsis’ (sterile) was necessary to make the cultures free from
micro-contamination. Haberlandt is thus regarded as the father of tissue culture. Efforts
continued to develop techniques for cultivation of plant cells under defined conditions.
Brilliant contributions came from R.J. Gautheret in France and P.R. White in U.S.A. in 1985.
Most of the modern tissue culture media have been derived from the work of Skoog and
coworkers during 1950-60.
Embryo Culture
Hannig [1904] initiated a new line of investigation involving the culture of embryogenic tissue.
He excised nearly mature embryos of some Crucifers and successfully grew them to maturity
on mineral salts and sugar solution. Winkler [1908] cultivated segments of string bean and
observed some cell divisions, but no proliferation. Embryo culture was also utilized by Laibach
in 1925 to recover hybrid progeny from an interspecific cross in Linum. Van Overbeek et al.
[1941] used coconut milk (embryo sac
fluid) for embryo development and callus formation in Datura which proved a turning point in
the field of embryo culture.
A new approach to tissue culture was conceived simultaneously by Kotte (Germany) and
Robbins (USA) in 1922. They postulated that a true in vitro culture could be made easier by
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using meristematic cells, such as those that operate in the root tip or bud. An important
breakthrough for continuously growing root tip cultures came from White (1934,1937), who
initially used yeast extract in a medium containing inorganic salts and sucrose but later
replaced it by three B vitamins, namely, pyridoxine, thiamine and nicotinic acid. White’s
synthetic medium later proved to be one of the basic media for a variety of cell and tissue
cultures.
Haploid Plants
Maheshwari and Guha (1964) of Botany Department, Delhi University were the first to produce
Haploid plants from anther culture of Datura. This marked the beginning of anther culture or
pollen culture for the production of haploid plants. The technique has been further developed
by Nitsch & Nitsch who isolated microspores of tobacco to produce complete plants.
Protoplasts Culture
What are Protoplasts?
Protoplasts are naked cells from which cell wall has been removed. In1960, Cocking produced
large quantities of protoplasts by using cell wall degrading enzymes. It is now
possible to regenerate whole plants from protoplasts and also to fuse protoplasts of different
species. In 1972, Carlson et al., produced the first somatic hybrid plant by fusing the
protoplasts of Nicotiana glauca and N. langsdorfii. Since then, many somatic hybrids have
been produced.
Let us now discuss the history of various hormones that are used in the tissue culture
media.
Role of Auxin
During mid-thirties, it was discovered that a successful establishment of callus cultures
depended on IAA (indole-3-acetic acid), the endogenous auxin and the role of B vitamins in
plant growth and in root cultures. Gautheret, White and Nobecourt in 1939, independently
established the first growing callus cultures from cambium tissue.
Gautheret (1934) cultured ‘cambium cells’ on the surface of a medium (Knop’s solution
containing glucose and cysteine hydrochloride) solidified with agar. After two months, he
observed proliferation of callus from these cells. He found that addition of ‘auxin’ (IAA-
indoleacetic acid) enhanced the proliferation of cambial cultures. White (1939) reported similar
results in the cultures from ‘tumour’ tissues of the hybrid Nicotiana glauca X N. langsdorfii.
Nobecourt also established continuously growing cultures of carrot slices. Finally, Gautheret,
White and Nobecourt gave the possibility of cultivating plant tissue for an unlimited period,
using media enriched with auxins.
Role of Cytokinin
Steward (1948) reported for carrot explants that ‘coconut milk’ enhanced more proliferation of
callus than did auxin. This indicated that the milk contained a stimulating substance that was
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not auxin. Skoog & Tsui (1951) demonstrated that ‘adenine’ stimulates cell division and
induces bud formation in tobacco tissue even in the presence of IAA (which normally acts as a
bud inhibitor). Skoog & Miller (1955) finally isolated a derivative of adenine (6- furfuryl
aminopurine) and named it as ‘Kinetin’. A substance with kinetin like properties was also
found in young maize endosperm which is called as Zeatin. It was also verified that a similar
substance called Ribosylzeatin occurred in coconut milk. Now, many synthetic as well as
natural compounds with kinetin- like activity are known which show bud- promoting
properties. These substances are collectively called ‘CYTOKININS’. These are used to induce
divisions in cells of highly mature and differentiated tissues (such as mesophyll or endosperm
from dried seeds), even in the presence of auxin in cultures.
Hormonal Control of Organ Formation
Before beginning with the study of plant tissue culture, it is necessary to understand the
mechanism of various hormones controlling the development of different organs in vitro. Skoog
& Miller (1957) proposed the concept of hormonal control of organ formation. They used
‘tobacco pith cultures’ and showed that root and bud initiation depends upon a balance
between auxin and kinetin. High concentration of auxin promoted ‘rooting’, whereas
proportionally more kinetin initiated bud or shoot formation. Unequal proportion of auxin and
cytokinin led to unorganized growth of the tissue. The determination of organogenesis also
depends upon the source of plant tissue, environmental factors, composition of media, polarity,
growth substances and other factors also, apart from hormonal balance only.
A General Idea about the Basic Terminology
Tissue culture is a good means for understanding the factors responsible for cell differentiation
and organ formation. In plant tissue culture experiments, we use either ‘single cell cultures’ or
‘explants’.
What is an ‘Explant’?
Explants are pieces of differentiated tissues which initiate growth in cultures. ‘Single cells’ can
be isolated either from cultured tissues or from intact plant organs. The explant can be
cultured on a medium to produce ‘callus’.
What is ‘Callus’?
Callus is an undifferentiated growth in solid form. It is an unorganized mass of cells. The
callus may be separated from explant and transferred to a fresh medium to get more tissue.
Pieces of undifferentiated calli are transferred to liquid medium, which is continuously agitated
to obtain a ‘Suspension Culture’. Agitation (shaking) of pieces breaks them into smaller clumps
and single cells and also maintains uniform distribution of cells and cell clumps in the
medium. It also allows gaseous exchange. Suspension cultures with single cells can also be
obtained from intact plant organs either
i. mechanically (grinding the tissue followed by cleaning, filteration and centrifugation)
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ii. enzymatically (treating excised and peeled leaves with macerozyme)- as in protoplast
isolation.
This is how single cells are isolated and maintained in liquid cultures. When an explant from
differentiated tissue is directly used for culture on a nutrient medium, the non- dividing,
dormant cells first undergo certain changes to achieve a meristematic state (dividing state).
The phenomenon of the reversion of mature cells to the meristematic state leading to the
formation of callus is called as ‘Dedifferentiation’.
Cells of the callus have the ability to form a whole plant, and this phenomenon is described as
‘Redifferentiation’. These two phenomenon of dedifferentiation and redifferentiation take place
due to ‘cellular totipotency’, found only in plant cells and not in animal cells. Therefore,
generally a callus phase is involved before the cells can undergo redifferentiation leading to
regeneration of a whole plant.
For more Definitions, follow the list given below:
Definitions for Plant Tissue Culture - Complete List
· Adventitious - in reference to a bud, embryo, root or shoot that arises in tissues and locations
that are not the normal origin in the plant.
· Aseptic - free from bacteria, fungi or other microorganisms.
· Autoclave - equipment that provides heat under high steam pressure for purposes of
sterilization.
· Axillary bud or shoot - a bud or shoot that arises from the axil of leaves or normal
origin.
· Callus - tissue that develops as a response to injury caused by physical or chemical
means; cells are differentiated but unorganized.
· Cell - a structural and physiological unit of a living organism (plant).
· DNA (Deoxyribonucleic acid) - composed of organic chemicals and is the genetic
material that ultimately determines an organism’s characteristics.
· Embryo - a rudimentary plant
· Epigenetic changes - persistent changes in phenotype that involve the expression of
particular genes.
· Explant - the plant part that is put into tissue culture.
· Gene - specific sequence of DNA that codes for a specific trait.
· Genotype - the sum total of all genes present in an organism (plant).
· Germ - cells or tissues that are involved in reproduction and have one-half of the genetic
material of somatic cells or tissues.
· Habituation - tissue cultures losing their requirements for a supply of exogenous
growth regulators.
· In vitro - isolated from the living organism and artificially maintained.
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· Micropropagation - the production of plants from plant parts used in tissue culture.
· Mutant - an organism (plant) that has a mutation.
· Mutation - change in the DNA sequence that is different from the original sequence.
· Organ - a distinct and visibly differentiated part of an organism.
· Organogenesis - refers to forming organs, in particular both roots and shoots from
callus cells.
· Phenotype - actual appearance and behavior of an organism (plant).
· Plantlets - small complete plants that were produced via tissue culture.
· Propagules - tissue that is divided and used for further multiplication.
· Somaclonal Variation - variation (usually in phenotype and/or perhaps genotype)
induced in cells by the tissue culture process.
· Somatic - cells or tissues that are vegetative and have the complete genetic material of
the organism (plant).
· Subculture - divide a propagule and transfer individual parts into other culture
vessels.
· Tissue - a group of cells organized into a structural and functional unit.
· Tissue culture - is the science of growing plant cells, tissues, or organs under
artificial conditions.
Types of Plant Tissue Culture
Plant tissue culture, which covers all types of aseptic plant culture, should be used in a
restricted sense and it is possible to distinguish it into various types of
cultures.
· Seed Culture - Culture of seeds in vitro to generate seedlings/plants
· Embryo Culture - Culture of isolated mature or immature embryos.
· Organ Culture - Culture of isolated plant organs. Different types can be distinguished,
e.g. meristem, shoot tip, root culture, anther tissue culture
· Callus Culture - Culture of a differentiated tissue from explant allowed to
dedifferentiate in vitro and a so-called callus tissue is produced.
· Cell culture - Culture of isolated cells or very small cell aggregates remaining
dispersed in liquid medium
· Protoplast culture - Culture of plant protoplasts, i,e., cells devoid of their cell walls.
· Anther culture - Culture of anthers.

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Types of Cultures
Introduction
Cultures are generally initiated from sterile pieces of a whole plant. These pieces are
termed ‘explants’ and may consist of pieces of organs, such as leaves or roots, or may be
specific cell types, such as pollen or endosperm. Many features of the explant are known to
affect the efficiency of culture initiation. Generally, younger, more rapidly growing tissue (or
tissue at an early stage of development) is most effective. Several different culture types most
commonly used will now be examined in more detail. This will give you a general idea about
the basic culture types used in tissue culture studies.

1. Callus Culture
What are the Characteristic Features of Callus?
Explants, when cultured on the appropriate medium, usually with both an auxin and a
cytokinin, can give rise to an unorganized, growing and dividing mass of cells called callus.
In culture, this proliferation can be maintained more or less indefinitely, provided that
the callus is subcultured on to fresh medium periodically. During callus formation there is
some degree of dedifferentiation (i.e. the changes that occur during development and
specialization are, to some extent, reversed), both in morphology (callus is usually composed of
unspecialized parenchyma cells) and metabolism (renewed and enhanced RNA and protein
syntheses). One major consequence of this dedifferentiation is that most plant cultures lose
the ability to photosynthesize. This has important consequences for the culture of callus tissue,
as the metabolic profile will probably not match that of the donor plant. This necessitates the
addition of other components such as vitamins and, most importantly, a carbon source to the
culture medium, in addition to the usual mineral nutrients.
Callus culture is often performed in the dark (the lack of photosynthetic capability being
no drawback) as light can encourage differentiation of the callus. During long-term culture, the
culture may lose the requirement for auxin and/or cytokinin. This process, known as
‘habituation’, is common in callus cultures from some plant species (such as sugar beet).
Applications of callus Culture
Callus cultures are extremely important in plant biotechnology. Manipulation of the
auxin to cytokinin ratio in the medium can lead to the development of shoots, roots or somatic
embryos from which whole plants can subsequently be produced. Callus cultures can also be
used to initiate cell suspensions, which are used in a variety of ways in plant transformation
studies.

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 2. Cell-Suspension Cultures
Callus cultures, broadly speaking, fall into one of two categories:
a) Compact or
b) Friable.
In compact callus the cells are densely aggregated, whereas in friable callus the cells are
only loosely associated with each other and the callus become soft and breaks apart easily.
Friable callus provides the inoculum to form cell-suspension cultures. The friability of the
callus can also sometimes be improved by culturing it on ‘semisolid’ medium (medium with a
low concentration of gelling agent). When friable callus is placed into a liquid medium (usually
the same composition as the solid medium used for the callus culture) and then agitated,
single cells and/or small clumps of cells are released into the medium. Under the correct
conditions, these released cells continue to grow and divide, eventually producing a cell-
suspension culture.
Liquid cultures must be constantly agitated, generally by a gyratory shaker at 100-250
rpm (revolution per minute), to facilitate aeration and dissociation of cell clumps into smaller
pieces. Suspension cultures grow much faster than callus cultures, need to be subcultured
about every week, allow a more accurate determination of the nutritional requirements of cells
and are the only system amenable to scaling up for a large scale production of cells and even
somatic embryos (SEs).
UNIT II
3. Immobilized Cell Cultures.
Plant cells and cell groups may be encapsulated in a suitable material, e.g., agarose and
Morris, calcium alginate gels, or entrapped in membranes or stainless steel screens. The gel
beads containing cells may be packed in a suitable column or, alternatively, cells may be
packed in a column of a membrane or wire cloth. Liquid medium is continuously run through
the column to provide nutrients and aeration to cells. Immobilization of cells changes their
cellular physiology in comparison to suspension culture cells; this offers several advantages for
their use in biochemical production, but they are usually not used for other studies.

What do you understand by Subculturing?

After a period of time, it becomes necessary, chiefly due to nutrient depletion and
medium drying, to transfer organs and tissues to fresh media. This is particularly true of
tissue and cell cultures where a portion of tissue is used to inoculate new culture tubes or
flasks; this is known as subculturing. In general, callus cultures are subcultured every 4-6
weeks, while suspension cultures need subcultured every 3-14 days. Plant cell and tissue
cultures may be maintained indefinitely by serial subculturing.

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 In case of suspension cultures, subculturing should be done about or somewhat prior to
the time of their maximum growth. The inoculum volume should be 20-25% of the fresh
medium volume; in any case, the initial cell density of the fresh culture (just after inoculation)
should be 5 x 104 cells /ml or higher otherwise the cells may fail to divide.

Selection of a Suitable Medium.

A suitable medium may be devised for a new system in several ways. A simple approach
is to first test-several concentrations, e.g., 0, 0.5, 2.5, 5 and 10m mol 1-1 of an auxin and a
cytokinin to identify a suitable combination of the two. Now, first different auxins and then
different cytokinins available to the worker may be tested to identify the best of each. Using
these GRs, the different standard recipes may be evaluated. One may then check the 1/2, full
and even higher salt concentration of the selected medium as well as different (2-6%) sucrose
concentrations. A further refinement requires testing 1/2, full and 2x concentrations of
individual components of the selected recipe. Alternatively, the worker may evaluate the
various combinations of low, medium and high concentrations of four solutions, i.e., minerals,
organics, auxin and cytokinin, to arrive at a suitable recipe; De Fossard et al. (1974) have
provided a broad spectrum experiment for this purpose (see, Bhojwani and Razdan, 1983).

4. Root Cultures
Root cultures can be established in vitro from explants of the root tip of either primary
or lateral roots and can be cultured on fairly simple media. The growth of roots in vitro is
potentially unlimited, as roots are indeterminate organs. Although the establishment of root
cultures was one of the first achievements of modern plant tissue culture, they are not widely
used in plant transformation studies.

5. Shoot Tip and Meristem Culture

The tips of shoots (which contain the shoot apical meristem) can be cultured in vitro,
producing clumps of shoots from either axillary or adventitious buds. This method can be used
for clonal propagation.
Shoot meristem cultures are potential alternatives to the more commonly used methods
for cereal regeneration as they are less genotype-dependent and more efficient (seedlings can
be used as donor material).

6. Embryo Culture
Embryos can be used as explants to generate callus cultures or somatic embryos. Both
immature and mature embryos can be used as explants. Immature, embryo-derived
embryogenic callus is the most popular method of monocot plant regeneration.
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Conclusion
Establishment of single cell cultures provides an excellent opportunity to investigate the
properties and potentialities of plant cells. Such systems contribute to our understanding of
the interrelationships and complementary influences of cells in multicellular organisms. The
single cell systems have a great potential for crop improvement. Free cells in cultures permit
quick administration and withdrawal of diverse chemicals/ substances, thereby making them
easy targets for mutant selection. Moreover, the individual cells within a population of cultured
cells invariably show cytogenetical and metabolic variations depending on the stage of the
growth cycle and culture conditions. Therefore, it is essential to understand the various types
of cultures in vitro and also their growth patterns.

Concept of totipotency

Introduction
To understand the pattern of organ development in cultures, you should first
understand the basic concept of totipotency. Two concepts, plasticity and totipotency are
central to understanding cell culture and regeneration. Plants, due to their sessile nature and
long life span, have developed a greater ability to endure extreme conditions and predation
than have animals. Many of the processes involved in plant growth and development adapt to
environmental conditions. This plasticity allows plants to alter their metabolism; growth and
development to best suit their environment. Particularly important aspects of this adaptation,
as far as plant tissue culture and regeneration are concerned, are the abilities to initiate cell
division from almost any tissue of the plant and to regenerate lost organs or undergo different
developmental ways in response to particular stimuli.

What is Totipotency?
When plant cells and tissues are cultured in vitro, they generally exhibit a very high
degree of plasticity, which allows one type of tissue or organ to be initiated from another type.
In this way, whole plants can be subsequently regenerated.
This regeneration of whole organisms depends upon the concept that all plant cells can,
given the correct stimuli, express the total genetic potential of the parent plant. This
maintenance of genetic potential is called ‘totipotency’.
Organogenic differentiation is an outcome of the process of dedifferentiation followed by
redifferentiation of cells. Dedifferentiation favours unorganized cell growth and the resultant
developed callus has meristems randomly divided. Most of these meristems, if provided
10
appropriate in vitro conditions, would redifferentiate shoot buds and roots. Mostly, the whole
plant regeneration from cultured cells may either occur through shoot-bud differentiation or
somatic embryogenesis.
The totipotency of somatic cells, under natural conditions, can also be observed to some
extent during the vegetative reproduction of plant species. The cell(s) of stem, leaf and root
cuttings of several plants are able to directly differentiate shoots and roots. The earliest reports
on controlled organogenesis in vitro were by White 1939 who obtained shoots on callus of a
tobacco hybrid and by Nobecourt 1939, who observed root formation in carrot callus. The
finding of White was confirmed and extended by Skoog 1944, who showed that auxin could
stimulate rooting and inhibit shoot formation. Further studies established that a balanced
combination of auxin and cytokinin controls the root and shoot formation (Skoog and Miller,
1957).

Organogenesis in Tobacco (Nicotiana tabacum)

Organogenesis from tobacco pith callus is the classical example of how varying plant
growth regulator regimes can be used to manipulate the pattern of regeneration from plant
tissue cultures.
 When cultured on a medium containing both auxin and cytokinin, callus will proliferate.
 If the auxin to cytokinin ratio is increased, adventitious roots will form from the callus
by organogenesis.
 If the auxin to cytokinin ratio is decreased adventitious shoots will be formed.
 If the explants are cultured on medium containing only a cytokinin shoots can be
produced directly.
Tobacco plants can also be easily regenerated from tobacco leaf pieces. Leaves are cut
into approximately 1 cm squares with a sterile scalpel (avoiding large leaf veins and any
damaged areas). The leaf pieces are then transferred (right side up) to gelled MS medium
supplemented with 1mg /l BAP (a cytokinin) and 0.1mgl/1 NAA (an auxin). Over the next few
weeks, callus forms on the explants, particularly around the cut surfaces. After 3 to 5 weeks
shoots emerge directly from the explants or from callus derived from the explants. When these
shoots are about 1cm long they can be cut at the base and placed on to solid MS medium
without any plant growth regulators. The shoots will form roots and form plantlets that will
grow in this medium and can subsequently be transferred to soil.
So, the auxin to cytokinin ratio of the medium determines which developmental
pathway the regenerating tissue will take. It is usual to induce shoot formation by increasing
the cytokinin to auxin ratio of the culture medium. These shoots can then be rooted relatively
simply.

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Factors affecting Regeneration
Regeneration via de novo organogenesis is a complex multistage phenomenon. There are
many factors which influence the process. In general, organized development is successfully
achieved through proper selection of the explant, proper choice of the medium, a balanced
combination of plant growth regulators and the control of physical environment. There is no
common formulation for all or many materials.

The major factors affecting the process of regeneration are

 Source of Explant
The factors which influence the response of the explant in the culture are:
1. The organ that is to be served as tissue source
2. The physiological and ontogenic age of the organ
3. The season in which the explant is obtained
4. The size of the explant
5. The overall quality of the plant from which explants are taken.
Regeneration from any plant part can be achieved by proper combination of the factors.
Stem segments and apices, root segments, leaf pieces, petioles, inflorescence sections, flower
petals, ovular tissue, seedling parts (cotyledon, hypocotyl) and seed embryos have been used
as an explant. All these explants can give rise to organs and embryos directly or indirectly via
callus. As mentioned above a suitable explant is desirable for a given species for successful
regeneration, e.g., embryonic tissues as an explant for regeneration in cereals. Explants
consisting of actively dividing cells have been useful in initiating the cultures and subsequent
regeneration.

 Nutrient Media and Constituents

Following medium constituents influence the regeneration more profoundly
1. Inorganic macro- and micro-nutrients – mostly MS and B5 media used
2. Carbon (energy) source – generally, 2-4% sucrose used
3. Reduced nitrogen source
4. Plant growth regulators – auxins (2,4-D, IAA, NAA and IBA) and cytokinins (kinetin and BA)
5. Vitamins – Inositol, Nicotinic acid, Pyridoxine and Thiamine.

 Culture Environment
These include:
1. Physical form of the medium i.e. presence or absence of agar
2. The pH of the medium
3. Light quality and quantity
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4. Temperature
5. Relative humidity, and
6. The gaseous atmosphere within the vessel.

Mechanism of Organ Development

The condition to achieve organogenesis, optimal conditions of medium constituents is
determined empirically. This is essential for each species or even each cultivar specifically for
clonal propagation. It is apparent that the process of differentiation begins with the changes in
individual cells in response to external stimuli. The cells determined to divide and produce
callus are changed to produce an organized structure by cellular differentiation. The
morphogenetic signals set up the conditions, which allow competent cells to undergo their
internally controlled programme of differentiation. Such a programme results from selective
gene action with the subsequent cellular processes of DNA replication and translation. This
would result in biochemical and biophysical changes in the target cells, including a change in
the metabolic activity. This is followed by cytological and histological changes leading to visible
appearance of organs formation.
Shoot regeneration is markedly affected by the genotype of explant in that different
varieties of a given species show quite different frequencies of shoot regeneration. In alfalfa,
breeding and selection drastically increased regeneration ability. In wheat, callus growth and
regeneration ability are governed by genes called, tissue culture response (TCR) genes, which
have been mapped on specific chromosomes. There are sufficient evidences that the process of
differentiation begins at single cell level but this does not mean that all organs are produced
from single cell. Once the stimulus is set in, centers of meristematic activity are formed
surrounding the cell from which the process starts. The organs are developed from these
multicellular meristematic centers. Clusters of meristematic cells, called nodules or
meristemoids arise in areas that accumulate starch, which is believed to serve as an energy
source for shoot bud differentiation. Meristemoids may develop vascular elements inside them,
while their outside may be made of cambium like cells. Initially, the meristemoids may either
produce a root or shoot. In, general, roots originate from inside the meristemoids (endogenous
origin), while shoots develop from outside (exogenous origin), but in some cases shoots
originate endogenously.
Conclusion
Organogenic differentiation is an outcome of the process of dedifferentiation followed by
redifferentiation of cells. Dedifferentiation favors unorganized cell growth and the resultant
developed callus has meristems randomly divided. Most of these meristems, if provided
appropriate in vitro conditions, would redifferentiate shoot buds and roots (whole plant
regeneration). This establishes the totipotency of somatic cells to undergo regeneration.
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Somatic embryogenesis
Objective
To understand the process of a single cell or a group of cells initiating the developmental
pathway that leads to reproducible regeneration of non-zygotic embryos capable of germinating
to form complete plants (Somatic embryogenesis). Under natural conditions, this pathway is
not normally followed, but from tissue cultures somatic embryogenesis occurs most frequently
and as an alternative to organogenesis for regeneration of whole plants.
How are Somatic Embryos produced?
In somatic (asexual) embryogenesis, embryo-like structures, which can develop into
whole plants in a way analogous to zygotic embryos, are formed from somatic tissues. These
somatic embryos (SE) can be produced either directly or indirectly.
Let us now understand the two ways in which somatic embryos are generated.
1. In direct somatic embryogenesis, the embryo is formed directly from a cell or small
group of cells without the production of an intervening callus. Though common from
some tissues (usually reproductive tissues such as the nucellus, styles or pollen), direct
somatic embryogenesis is generally rare in comparison with indirect somatic
embryogenesis.
2. In indirect somatic embryogenesis, callus is first produced from the explant. Embryos
can then be produced from the callus tissue or from a cell suspension produced from
that callus.
Carrot is the classical example of indirect somatic embryogenesis and is
explained in more detail below:
Indirect somatic embryogenesis in carrot (Daucus carota):
A callus can be established from explants from a wide range of carrot tissues by placing
the explant on solid medium (e.g. Murashige and Skoog (MS)) containing 2, 4-D (1mg/l). This
callus can be used to produce a cell suspension by placing it in agitated liquid MS medium
containing 2, 4-D (1mg/l). This cell suspension can be maintained by repeated subculturing
into 2, 4-D-containing medium. Removal of the old 2, 4-D-containing medium and replacement
with fresh medium containing abscisic acid (0.025mg/l) results in the production of embryos.

Direct somatic embryogenesis from alfalfa (Medicago falcata)

Young trifoliate leaves are used as the explant. These are removed from the plant and
chopped into small pieces. The pieces are washed in a plant growth regulator-free medium and
placed in liquid medium (B5) supplemented with 2, 4-D (4mg/ 1), kinetin (0.2mg/1), adenine
(1mg/1) and glutathione.

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Developmental Patterns of SEs
Somatic embryogenesis usually proceeds in two distinct stages.
1. In the initial stage (embryo initiation), a high concentration of 2, 4-D is used.
2. In the second stage (embryo production) embryos are produced in a medium with no or
very low levels of 2,4-D.
In somatic (asexual) embryogenesis, embryo-like structures, which can develop into
whole plants in a way analogous to zygotic embryos, are formed from somatic tissues. SEs
generally originate from single cells which divide to form a group of meristematic cells. Usually,
this multicellular group becomes isolated by breaking cytoplasmic connections with the other
cells around it and subsequently by cutinization of the outer walls of this differentiating cell
mass. The cells of meristematic mass continue to divide to give rise to globular (round ball
shaped), heart- shaped, torpedo and cotyledonary stages.
In general, the essential features of SE development, especially after the globular stage,
are comparable to those of zygotic embryos. Somatic embryos are bipolar structures in that
they have a radicle and a plumule. The radicular end is always oriented towards the center of
callus or cell mass, while the plumular end always sticks out from the cell mass. In contrast, a
shoot bud is monopolar as it does not have a radicular end. In many SEs, radicle is
suppressed so that they often do not produce roots; in such cases, roots have to be
regenerated from the shoots produced by germinating SEs. SEs often show abnormal
developmental features, e.g., 3 or more cotyledons, bell- shaped cotyledon, larger size etc.
These problems are often overcome by the presence of ABA or a suitable concentration of
mannitol.
In some species, normal looking somatic embryos are formed but they fail to germinate.
The SE regenerating from explant or callus is termed as primary somatic embryo. In many
cases, SEs regenerate from the tissues of other SEs or the parts of germinating SEs; such SEs
are called secondary somatic embryos.
Somatic embryogenesis is influenced by several factors:
1. Growth Regulators
In most species an auxin (generally 2, 4-D at 0.5- 5mg/l) is essential for somatic
embryogenesis. The auxin causes dedifferentiation of a proportion of cells of the explant which
begin to divide. The ability to regenerate SEs, i.e., totipotency, is acquired by cells during
dedifferentiation in response to high auxin treatment. High auxin prevents its own polar
transport. 2, 4-D is the most commonly used auxin in somatic embryogenesis, the others are
2,4,5-T, picloram, Dicamba etc.
There are exceptional cases like carrot, where auxin is not required for somatic
embryogenesis and its presence can inhibit the process. Auxins promote hypermethylation of
DNA which may have a role in totipotency acquisition.
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Somatic embryogenesis is a two step process:
1. SE induction on a high auxin (upto 40-60mg/l 2,4-D)
2. SE development on a low auxin or GR- free medium

2. Nirogen Source
The form of nitrogen has a marked effect on somatic embryogenesis. In carrot, NH4+ has
a promotive effect on SE regeneration. In fact, induction of SEs in carrot occurs only when
about 5 m mol/kg of cell fresh weight NH4+ is present in the cells. This level of endogenous
NH4+ is reached with only 2.5m mol/l of exogenous level of NH4+, while 60m mol/l NO3- is
needed for the same. Therefore, the presence of a low level of NH4+ (in carrot 10m mol/l is
optimal) in combination with NO3- is required for SE regeneration. In carrot, NH4+ is essential
during SE induction, while SE development occurs on a medium containing NO3- as the sole
nitrogen source.

3. Genotype of the Explant:

Explant genotype has a marked influence on SE regeneration. Strong genotypic effects
have been shown in many species, e.g., alfalfa, wheat, maize, rice, chickpea etc. where
individual genes affecting SE regeneration have been identified. Variation for regeneration
ability is mainly additive and highly heritable in maize, rice and wheat, but in barley,
dominance seems to be more important. In case of wheat, rice and maize, cytoplasm has a
strong influence on regeneration. It may be postulated that at least a part of the genotypic
effect on regeneration may be concerned with endogenous GR levels and/or sensitivity to
exogenous GRs.

4. Other Factors
Certain other factors, such as high K+ levels and low dissolved Oxygen levels promote
SE regeneration in some species. In some other species, e.g., Citrus medica, some volatile
compounds like ethanol inhibit SE regeneration. In soyabean, low sucrose concentrations (5
and 10 g/l) promote SE regeneration as compared to high concentrations (20 and 30 g/l). In
alfalfa, use of maltose as carbon source improves both SE induction and maturation (including
germination) as compared to those on sucrose.
Isolation of protoplasts:
Mechanical isolation is done by cutting plasmolysed tissue with a sharp edged knife and
releasing the protoplasts by deplasmolysis. The principal deficiency of this approach is that the
protoplasts released are few in number; mechanical isolation is thus only of historical
importance now. Isolation of protoplasts mechanically from higher plants was pioneered by
Klercker in 1892. Generally, protoplasts were isolated from highly vacuolated cells of storage
tissues (onion bulbs, scales, radish root, mesocarp of cucumber and beet root).

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