Download Concepts of Micropropagation

• Specific terms are used to describe the sexual expression of
individual plants within a population.
• Hermaphrodite - A plant that has only bisexual reproductive units
(flowers, conifer cones, or functionally equivalent structures).
• Monoecious - an individual that has both male and female
reproductive units (flowers, conifer cones, or functionally
equivalent structures) on the same plant; from Greek for "one
household".
• Individuals bearing separate flowers of both sexes at the same time
are called simultaneously or synchronously monoecious.
• Protoandrous describes individuals that function first as males and
then change to females; protogynous describes individuals that
function first as females and then change to males.
• Dioecious - refers to a plant population having separate male and
female plants. That is, no individual plant of the population
produces both microgametophytes (pollen) and megagametophytes
(ovules); individual plants are either male or female. From Greek for
"two households". [Individual plants are not called dioecious; they
are either gynoecious (female plants) or androecious (male plants).]
• In other words, Individual species which have separate male and
female plants, and are either one sex or the other, are called
dioecious
– Androecious - plants producing male flowers only, produce pollen but
no seeds, the male plants of a dioecious population.
– Gynoecious - plants producing female flowers only, produces seeds
but no pollen, the female of a dioecious population. In some plant
populations, all individuals are gynoecious with non sexual
reproduction used to produce the next generation.
• Subdioecious, a tendency in some dioecious populations to
produce monoecious plants.
• The population produces normally male or female plants but some
are hermaphroditic, with female plants producing some male or
hermaphroditic flowers or vice versa.
• The condition is thought to represent a transition between
hermaphroditism and dioecy..
– Gynomonoecious - has both hermaphrodite and female structures.
– Andromonoecious - has both hermaphrodite and male structures.
– Subandroecious - plant has mostly male flowers, with a few female or
hermaphrodite flowers.
– Subgynoecious - plant has mostly female flowers, with a few male or
hermaphrodite flowers.
• Polygamy - Plants with male, female and
perfect (hermaphrodite) flowers on the same
plant, called trimonoecious or
polygamomonoecious plants.
• Diclinous , an angiosperm term, includes all
species with unisexual flowers, although
particularly those with only unisexual flowers,
i.e. the monoecious and dioecious species.







Ornamental plants or garden plants are typically
grown in the flower garden or as house plants.
Most commonly they are grown for the display of
their flowers.
Other common ornamental features include leaves,
scent, fruit, stem and bark.
In some cases, unusual features may be considered
ornamental, such as the prominent and rather vicious
thorns of Rosa sericea.
In all cases, their purpose is the enjoyment of
gardeners and visitors.
Ornamental plants may also be used for landscaping,
and for cut flowers.
The adequate spacing between pots of plants
prevents them from competing for sunlight.
 Similarly
trees may be called ornamental
trees.
 This term is used when they are used as part
of a garden setting, for instance for their
flowers, their shapes or for other attractive
characteristics.
 By comparison, trees used in larger
landscape effects such as screening and
shading, or in urban and roadside plantings,
are called amenity trees.
For plants to be considered as ornamental, they
may require specific work and activity by a
gardener.
 For instance, many plants cultivated for topiary
and bonsai would only be considered as
ornamental by virtue of the regular pruning
carried out on them by the gardener, and they
may rapidly cease to be ornamental if the work
was abandoned.
 Pruning is the process of removing certain
above-ground elements from a plant; in
landscaping this process usually involves removal
of diseased, non-productive, or otherwise
unwanted portions from a plant.





Ornamental plants and trees are distinguished
from utilitarian and crop plants, such as those
used for agriculture and vegetable crops, and for
forestry or as fruit trees.
This does not preclude any particular type of
plant being grown both for ornamental qualities
in the garden, and for utilitarian purposes in
other settings.
Thus lavender is typically grown as an
ornamental plant in gardens, but may also be
grown as a crop plant for the production of
lavender oil.
Other types of ornamental plants include the lily,
rose, morning glory and the pink oak.
 Simple
sterile cultures for micropropagation
of ornamentals using direct application of
chlorine disinfectants by preparing sterile
medium without autoclaving and inoculating
explants without the laminar air-flow cabinet
were developed.
These techniques could be applied to various
micropropagation processes of ornamentals.
 The sterile medium could be prepared
without autoclaving by immediately
incorporating chlorine disinfectants into the
medium.
 In these cases, all chlorine disinfectants
tested were effective for sterile medium
preparation.

The media could be used for sterile cultures
in various micropropagation processes.
 Spraying the surface of a medium and the
whole explants with chlorine disinfectants
after inoculating was effective for
inoculating explants sterilely and for
subsequent sterile cultures under nonsterilized conditions.
 These techniques could be applied to the
following cultures, shoot tips of
chrysanthemum and Cymbidium, stem
section explants of chrysanthemum.

The treated levels of incorporated and
sprayed chlorine disinfectants suppressed in
vitro contamination and did not appear to be
toxic to shoots or plantlets of ornamentals
tested.
 Propagated plantlets which were cultured on
the disinfectant incorporated medium and
handled with spraying treatments under nonsterile conditions could survive without
harming tissues and were raised without in
vitro contamination.

Horticulture is the industry and science of
plant cultivation including the process of
preparing soil for the planting of seeds,
tubers, or cuttings.
 Horticulturists work and conduct research in
the disciplines of plant propagation and
cultivation, crop production, plant breeding
and genetic engineering, plant biochemistry,
and plant physiology.





The work particularly involves fruits, berries,
nuts, vegetables, flowers, trees and shrubs.
Horticulturists work to improve crop yield,
quality, nutritional value, and resistance to
insects, diseases, and environmental stresses.
Horticulture usually refers to gardening on a
smaller scale, while agriculture refers to the
large-scale cultivation of crops.
The word is composite, from two words, horti,
meaning grass, originating in the Greek meaning
the same (grass) and the word culture.
Propogation is by suckers or off-shoots which
spring at the base of a banana-tree from
underground rhizomes.
 Vigorous suckers, with stout base, tapering
towards the top and possessing narrow
leaves, are selected for plant.
 Each sucker should have a piece of
underground stem with a few roots attached
to it.

Banana suckers can be planted throughout
the year in southern India, except during
summer, whereas in the rest of the country,
the rainy season is preferred.
 They are planted in small pits, each just
enough to accommodate the base of a
sucker.
 The planting-distance varies from 2m X 2m in
the case of dwarf varieties to 4m X 4m in the
case of very tall varieties.







An application of 20 to 25 kg of farmyard manure, together
with about 5 kg of wood-ashes per plant is given at
planting time.
In southern India, ammonium sulphate is applied one
month, five months and nine months after planting 20 kg
per hectare each time.
In western India, a little over 2 kg of oilcake per stool is
applied during the first three months after planting.
A complete fertilizer mixture may be applied to supply 100
to 200 kg of N, 100 to 200 kg of P2O5 and 200 to 400 kg of
K2O per hectare.
A green-manure crop is also considered beneficial.
Trials at the Indian Institute of Horticultural Research have
shown that for the 'Robusta' variety, a fertilizer mixture
comprising 180 g of N + 108 g of P2O5 + 225 g of K2O per
plant is ideal. (phospjorous pentoxide+potassium peroxide
mixture).
 The
banana and mango-plants require very
heavy irrigation.
 Irrigation is given in most places once in
seven to ten days.
 Stagnation of water in the soils is not very
congenial to the proper growth of banana
and, hence, the drainage of soil is also
essential.
Early varieties commence flowering in southern
and western India about seven months after
planting, and the fruits take about three months
more to ripen.
 In the Andhra Pradesh delta areas, the fruits are
ready for harvesting about seven to eight months
after planting.
 The first crop of the 'Poovan' variety matures in
12 to 14 months and the second in 21 to 24
months after planting.
 In other parts of India, the first crop is usually
gathered a year after planting, whereas the
succeeding crop may be ready in six to ten
months thereafter.

 No
systematic pruning is done.
 The removal of dead-wood and the thinning
of over-crowded and mis-shapen branches
after about four years is all that is necessary;
flowers that appear during the first three or
four years should be removed.






The ripening of banana or mango is done in several
ways, e.g. exposing the bunches to the sun, placing
them over a hearth, wrapping them in closed
godowns or smoking them in various ways.
One of the common ways is to heap the fruits in a
room and cover them with leaves, after which fire is
lit in a corner and the room is closed and made as
air-tight as possible.
Ripening takes place usually in 30 to 48 hours.
In a cool store, the bunches ripen well at about 15o
to 20oC.
The application of vaseline, a layer of clay or coaltar to the cut-ends of the stalks prevents rotting
during ripening and storage.
Wrapping up the fruits and packing them in crates
help to reduce the damage during transport.
 In
animal or plant development,
organogenesis (organo-genesis, compound of
the Greek words "that with which one
works", and "origin, creation, generation") is
the process by which the ectoderm,
endoderm, and mesoderm develop into the
internal organs of the organism.









Organogensis refers to that period of time during development when the organs
are being formed. After an egg has been fertilized, and has been implanted in the
uterus, the developing form is known as the embryo.
Organogenesis takes place during this embryonic phase.
In fact, most organogenesis has begun as early as week five in humans
(remember that a normal human pregnancy lasts an average of 40 weeks).
Therefore, damage to any of the organ systems of the body which may ultimately
result in some type of birth defect usually strikes during this time frame.
By week five, the buds of tissue which will become the limbs are in place.
The structures which will become the skeleton, nervous system, and circulatory
system of the face, neck, and jaws are in place.
A five-week-old embryo has the early developmental structures of the esophagus,
stomach, intestine, liver, and pancreas.
The heart is already functioning, and continues to develop and change over this
period of time. The respiratory system begins developing, as do blood vessels,
blood cells, nervous and endocrine organs.
Clearly, the most crucial organs of the human form are developing during
organogenesis. Essentially, the earlier the injury to these developing buds of
tissue, the more severe the ultimate defect. This is because these tiny buds of
tissue hold all the primitive cells which should differentiate into the myriad
number of cells necessary to create all of the varied organs of the human body.
 It is an irony that, during this crucial period of
development, when toxins from the outside world
can have such devastating effects on the ultimate
development of the embryo, many women are not
even yet aware that they are pregnant, and are
therefore not in the mindframe of protecting the
developing embryo from exposure to such harmful
substances as cigarette smoke, alcohol, certain
drugs or medications, or extremes of heat .
Embryo Rescue
•
•
•
•
•
•
•
One of the most infamous agents (teratogens) responsible for widespread
deformities during the period of organogenesis is a drug called thalidomide.
Thalidomide was administered to women (particularly in Europe in the 1950s)
because it was thought to combat the nausea present in early pregnancy.
Over time, however, it became evident that babies born of thalidomide-using
mothers had very high rates of serious limb deformities.
In particular, the long bones of the limbs were either absent or seriously
deformed.
Furthermore, many of these children had associated defects of the heart and
intestine.
Thalidomide was ultimately determined to be at fault, causing the most severe
defects when given between weeks four and six of pregnancy: the period of
organo-genesis.
Thalidomide was subsequently withdrawn from the market.
 Plant
embryogenesis is the process that
produces a plant embryo from a fertilised
ovule by asymmetric cell division and the
differentiation of undifferentiated cells into
tissues and organs.
 It occurs during seed development, when the
single-celled zygote undergoes a programed
pattern of cell division resulting in a mature
embryo.
 A similar process continues during the plant's
life within the meristems of the stems and
roots.




Embryogenesis occurs naturally as a result of sexual
fertilization and the formation of the zygotic embryos.
The embryo along with other cells from the motherplant
develops into the seed or the next generation, which, after
germination, grows into a new plant.
Embryogenesis may be divided up into two phases, the
first involves morphogenetic events which form the basic
cellular pattern for the development of the shoot-root
body and the primary tissue layers; it also programs the
regions of meristematic tissue formation.
The second phase, or postembryonic development,
involves the maturation of cells, which involves cell growth
and the storage of macromolecules (such as oils, starches
and proteins) required as a 'food and energy supply' during
germination and seedling growth.




Embryogenesis involves cell growth and division,
cell differentiation and programed cellular
death.
The zygotic embryo is formed following double
fertilisation of the ovule, giving rise to two
distinct structures: the plant embryo and the
endosperm which together go on to develop into
a seed.
Seeds may also develop without fertilization,
which is referred to as apomixis.
Plant cells can also be induced to form embryos
in plant tissue culture; such embryos are called
somatic embryos.




Following fertilization, the zygote undergoes an
asymmetrical cell division that gives rise to a small apical
cell, which becomes the embryo and a large basal cell
(called the suspensor), which functions to provide
nutrients from the endosperm to the growing embryo.
From the eight cell stage (octant) onwards, the zygotic
embryo shows clear embryo patterning, which forms the
main axis of polarity, and the linear formation of future
structures.
These structures include the shoot meristem, cotyledons,
hypocotyl, and the root and root meristem: they arise
from specific groups of cells as the young embryo divides
and their formation has been shown to be positiondependent.
In the globular stage, the embryo develops radial
patterning through a series of cell divisions, with the outer
layer of cells differentiating into the 'protoderm.'







Embryonic tissue is made up of actively growing cells and the term is
normally used to describe the early formation of tissue in the first stages
of growth.
It can refer to different stages of the sporophyte and gametophyte plant;
including the growth of embryos in seedlings, and to meristematic
tissues, which are in a persistently embryonic state, to the growth of new
buds on stems.
In both gymnosperms and angiosperms, the young plant contained in the
seed, begins as a developing egg-cell formed after fertilization
(sometimes without fertilization in a process called apomixis) and
becomes a plant embryo.
This embryonic condition also occurs in the buds that form on stems.
The buds have tissue that has differentiated but not grown into
complete structures.
They can be in a resting state, lying dormant over winter or when
conditions are dry, and then commence growth when conditions become
suitable.
Before they start growing into stem, leaves, or flowers, the buds are
said to be in an embryonic state.







Somatic embryos are formed from plant cells that are not normally
involved in the development of embryos, i.e. ordinary plant tissue.
No endosperm or seed coat is formed around a somatic embryo.
Applications of this process include: clonal propagation of genetically
uniform plant material; elimination of viruses; provision of source tissue
for genetic transformation; generation of whole plants from single cells
called protoplasts; development of synthetic seed technology.
Cells derived from competent source tissue are cultured to form an
undifferentiated mass of cells called a callus.
PGR’s in the tissue culture medium can be manipulated to induce callus
formation and subsequently changed to induce embryos to form from the
callus.
The ratio of different PGR’s required to induce callus or embryo
formation varies with the type of plant.
Asymmetrical cell division also seems to be important in the development
of somatic embryos, and while failure to form the suspensor cell is lethal
to zygotic embryos, it is not lethal for somatic embryos.




Somatic embryogenesis refers to the remarkable ability of
nonzygotic plant cells (including haploid cells) to develop
through characteristic embryological stages into an embryo
capable of developing into a mature plant.
Somatic embryogenesis is an expression of totipotency and
the associated differential gene expression.
Somatic embryos may be produced in nature in certain
plant species as a form of apomixis known as adventitious
embryony.
Somatic embryogenesis in plants usually refers to the
induction of somatic embryos in vitro, first demonstrated
by both Steward and Reinert in 1958.
Research into somatic embryogenesis has intensified as
plant regeneration in vitro has come to be widely utilized
in transformation and somatic hybridization.
Artificial Seeds- An artificial seed is a bead of
gel containing a somatic embryo (or shoot bud),
and the nutrients, growth regulators pesticides,
antibiotics, etc. needed for the development of
a complete plantlet from the enclosed SE/shoot
bud.
 Synthetic seeds or artificial seeds are the livingseed like structure derived from somatic
embryoids (or, somatic embryos) in-vitro culture
after encapsulation by a hydrogel.
 The preserved embryoids are called synthetic
seeds.

Artificial seeds
 The use of somatic embryos as artificial seeds for large scale clonal
propagation of plants is close to becoming a reality.
 The quality of the artificial seed depends on the temporal,
quantitative and qualitative supply of growth regulator and
nutrients along with an optimal physical environment.
 Desiccation of somatic embryos provides a quiescent phase
analogous to true seeds, facilitating the convenience of year round
production, storage and distribution.
 Somatic embryos possess the ability to express desiccation
tolerance in response to an external chemical or physical stimuli.
 The mechanisms of desiccation tolerance involve stabilization of
membranes in dry state and prevention of oxidative degradation of
biomolecules. Encapsulation of embryo may control the water
uptake, release of nutrients and provide mechanical protection
required for field planting.
 Artificial
seeds may be produced using one of
the following two ways:
(1) Desiccated systems and
(2) Hydrated systems.
In the desiccated system, SE s is first
hardened to withstand desiccation and then
are encapsulated in a suitable coating
material to yield desiccated artificial seeds.
 SE s may be hardened either by
treating/coating mature SE s with a suitable
polymer followed by drying, or treating them
with ABA during their maturation phase.
 ABA treatment also improves germination of
SE s, and is used even in the hydrated
systems.

In the hydrated systems, SE s are enclosed in
gels, which remain hydrated.
 Of the many gels evaluated, calcium alginate
is the most suitable.
 Artificial seeds can be easily made as
follows. A 2% solution of sodium alginate is
filled in a burette and allowed to drip drop
by drop into a 100 mM CaCl2 solution.






As the sodium alginate bead or drop forms at the tip of the burette, an
SE is inserted into it with the help of a spatula before the drop falls into
the CaCl solution.
The beads become hardened as calcium alginate is formed; after about
20-30 min the artificial seeds are removed, washed with water and used
for planting.
Hydrated artificial seeds are sticky and difficult to handle on a large
scale, and dry rapidly in the open air.
These problems can be resolved by providing a waxy coating over the
beads.
Alternatively, a desiccated system may be used to produce artificial
seeds.
However, hydrated artificial seeds have to be planted soon after they are
produced. Precision machines for a large scale encapsulation of SE s have
been devised.
General Procedure
The general procedure is as followed:
1) Induction of somatic embryogenesis
2) Maturation of somatic embryos
3) Encapsulation of somatic embryos (synthetic seeds)
4) Evaluation of embryoid and plant conversion
5) Planting in fields/green house
Advantages
1) It could be preserved for long time at lower temperature
2) Rooting, hardening and conversion steps are waved off as
these seeds can directly be sowed in the fields like natural
seeds
1)
2)
3)
4)
5)
6)
For the development of hybrid plants which have
unstable genotypes or show seed sterility.
In case that the seeds become sterile, the
immature embryo from the seed can be rescued
(called as embryo rescue) and then can be
encapsulated artificially with appropriate growth
medium to allow its maturation and desiccation for
its germination.
Germplasm conservation.
For various research and analysis purposes like
studying the role of endosperm, etc.
To produce large number of the clones of elite
species at the cheaper cost.
To supply adjuvant like plant growth regulators,
pesticides, etc.
Anther Culture
• Anter culture is the process of using anthers to culture haploid
plantlets.
• The technique was discovered in 1964 by Guha and Maheshwari.
• This technique can be used in over 200 species, including tomato,
rice, tobacco, barley, and geranium.
• Some of the advantages which make this a valuable method for
obtaining haploid plants are:
 The technique is fairly simple
 It is easy to induce cell division in the immature pollen cells in some
species
 A large proportion of the anthers used in culture respond (induction
frequency is high)
 Haploids can be produced in large numbers very quickly.
In experiments using Datura innoxia,
induction frequencies of almost 100% and a
yield of more than one thousand plantlets or
calluses have occurred under optimal
conditions from one anther.
 Success can be determined within 24 hours
as cells begin to divide.

Some disadvantages of using anther culture to
obtain haploids are:
1) When working with some species, the majority
of plants produced have been non-haploid .
2) In cereals, very few green plants are obtained;
many of the plants are albinos or green-albino
chimeras. (a chimera is an animal that has
two or more different populations of
genetically distinct cells that originated in
different zygotes; )
3) It is tedious to remove the anthers without
causing damage.
4) Sometimes a particular orientation is necessary
to acheive a desired response.
This diagram shows the various stages of anther
and isolated pollen culture. The stages of anther
culture from anther to haploid plantlet can be
described as follows:
 a) an unopened flower bud,
 1b) anthers,
 1c) the anthers in culture,
 1d) and
 1e) proliferating anther,
 1f) haploid callus,
 1g) differentiating callus,
 h) haploid plantlet.

Isolated pollen culture is as follows:
 a) an unopened flower bud,
 3b) isolated pollen from a cultured anther,
 3c) pollen culture,
 3d) multinucleate pollen,
 3e) and
 3f) pollen embryo.
 Homozygous plants can be obtained by
treating the haploid plantlets with
colchicine.

Colchicine is a toxic natural product and
secondary metabolite, originally extracted
from plants of the genus Colchicum and used
originally to treat rheumatic complaints.
 Colchicine's present medicinal use is in the
treatment of gout and familial Mediterranean
fever.
 It is also being investigated for its use as an
anti-cancer drug.
 In neurons, axoplasmic transport is
disrupted by colchicine.

Protocol for Pollen Culture
1. Flower buds are collected from the plants after all preparations
for culturing in the laboratory have been made.
2. Buds are collected in a nonsterile petri dish and the length of
each bud measured using a cm scale. Buds of corolla length 21-23
mm are chosen as the pollen in them would have usually completed
the first mitosis.
3. The buds are chilled for 12 days (7 to 8°C) in a refrigeration unit.
4. They are then surface sterilized in a petri dish containing a
suitable sterilizing agent, for instance 0.01 % solution of mercuric
chloride.
• 5. Using sterile, double distilled water, the buds are rinsed 3-4 times
in a sterile air cabinet.
6. Using forceps and a dissecting needle, the buds are carefully
teased open and the anthers removed.
7. The anthers dissected out from each bud are grouped separately
on removal.
8. One anther from each group is then removed and squashed in
acetocarmine stain in order to determine the stage of pollen
development. The pollen should be in the first pollen division.
9. For each culture, anthers from tobacco buds are placed in 5 ml of
liquid medium in a petri dish.
10. After regular intervals of 6, 10 and 14 days of
culture, the anthers are removed from the culture and
discarded. That ensures the dehiscence of all anthers and
release of pollen into the culture medium.
11. The dishes are then sealed with parafilm and incubated
at 28°C in darkness for the first 14 days of culture.
12. After 14 days, the cultures are transferred to an
illuminated growth chamber (500 lux, 12-h daylength,
25°C).
13. The growth of haploid embryos to plantlets developed
from released pollen should be observed.
14. Follow steps 13 and 14 given in the protocol for anther
culture for growing the plantlets to maturity.
These are cultured primarily for the
production of Haploid plants which find
important application in the field of plant
breeding.

Pollen culture is also termed as
Microspore Culture.

Haploids are sporophytes of higher plants
with gametophytic chromosome constitution.

It is possible to induce haploidy either
using anther culture, henceforth reffered to
as ‘androgenesis’ or from cultures of
individual pollen grains.

Pollen culture offers certain advantages
over anther culture due to the elimination of
anther wall, e.g.,
 i. Studies on differentiation and development
are easier and more precise,
 ii. No callus formation can occur from wall
tissue and
 iii. Products from different pollen grains
ordinarily do not get mixed up (this
eliminates the risk of chimera).
 A).
Culture Medium
Medium requirements may vary with:
 1. Species,
 2. Genotype,
 3. Age of donor plants and anthers, and
 4. Conditions under which the donor plants
are grown.
For example, pollen grains of Datura and
tobacco produce embryos on an agar medium
containing only 2-4% sucrose.
 For most plant species, a complete tissue
culture
medium is required.
 MS, LS (Linsmaer and Skoog) and some
other tissue culture media are generally
used.
 Media with dilute salt solutions, e.g.,
White’s and Heller’s media, are ordinarily
supplemented with coconut milk.

 B).
Growth Regulators
 C). Stage of Pollen Development
 etc., the optimum stage is just before or just
after the first pollen mitosis, while the early
binucleate stage is the most suitable for
species



Anther cultures are generally maintained in
alternating periods of light (12-18 hr; 5,00010,000 lux m2) at 28°C and darkness (12-16 hr)
at 22°C, but the optimum conditions vary with
the species.
The walls of responsive anthers turn brown and
after 3-8 weeks they burst open due to the
developing callus or embryos.
After the seedlings (from embryos) or shoots
(from callus) become 3-5 cm long, they are
transferred to a medium conducive to good root
development.
Exposure of excised flower buds to a low
temperature for some time e.g., at 3-5°C for
2 days or at 7-8°C for 12 days for tobacco,
prior to removal of anthers for culture may
markedly enhance the recovery of haploid
plants.
 In some species, however, a brief exposure
of anthers to a high temperature is reported
to have a promotory effect.





Androgenesis in barley is promoted by the use of
wheat or barley starch as gelling agent (in place of
agar), and by the addition of ficoll (a neutral, highly
branched, high-mass, hydrophilic polysaccharide
which dissolves readily in aqueous solutions)in liquid
medium.
In many species, activated charcoal (in agar-gelled
media) is promotive.
In addition, amino acids like glutamine, proline,
serine etc. enhance the frequency of responsive
anthers.
Anther extracts and media conditioned byculturing
anthers for few days improve androgenesis; thus
anther wall seems to provide some nutritional
factors.






Somaclonal variation It is the term used to describe the
variation seen in plants that have been produced by plant
tissue culture.
Chromosomal rearrangements are an important source of
this variation.
Somaclonal variation is not restricted to, but is particularly
common in plants regenerated from callus.
The variations can be genotypic or phenotypic, which in
the later case can be either genetic or epigenetic in origin.
Typical genetic alterations are: changes in chromosome
numbers (polyploidy and aneuploidy), chromosome
structure (translocations, deletions, insertions and
duplications) and DNA sequence (base mutations).
Typical epigenetic related events are: gene amplification
and gene methylation
Historically, plant cell culture has been viewed by
most to be a method for rapid cloning.
 In essence, it was seen as a method of sophisticated
asexual propagation, rather than a technique to add
new variability to the existing population.
ORIGINS AND MECHANISMS OF SOMACLONAL
VARIABILITY:
 Somaclonal variation can be of two sorts:
1)Genetic (i.e. heritable) variability – caused by
mutations or other changes in DNA.
2)Epigenetic (i.e. non-heritable) variability – caused
by temporary phenotypic changes.
 Various molecular mechanisms are responsible for
genetic variability associated with somaclonal
variation.

One of the more frequently encountered
types of somaclonal variation results from
changes in chromosome number, that is,
aneuploidy, polyploidy, or mixoploidy.
 Changes in ploidy originate from
abnormalities that occur during mitosis.
 For example, extra chromosomal duplication
during interphase, spindle fusion or lack of
spindle formation and cytoplasmic division.
 A plant cell grows and age, the frequency of
changes in ploidy increases.









Therefore, changes in ploidy observed in cultures and regenerated
plants might have their origins in the source of tissue explants used.
Another cause of variability due to changes in ploidy is the in vitro
culture regime itself.
The longer the cell remains in culture the greater is its chromosomal
instability.
In addition, the composition of the growth medium can trigger changes
in ploidy.
For example, both kinetin and 2, 4-D are implicated in ploidy changes
and cultures grown under nutrient limitation can develop abnormalities.
Selecting a suitable explant and an appropriate culture medium can
therefore enhance the chromosomal stability of the culture.
However, high variations of ploidy in cultures do not always lead to high
frequencies of somaclonal variation in regenerated plants.
This is because, in mixed cultures, diploid cells appear to be better
fitted than aneuploid or polyploidy cells for regeneration, as they are
more likely to form meristems.
 Structural
changes in nuclear DNA appear to
be a major cause of somaclonal variation.
The changes can modify large regions of a
chromosome and so may affect one or
several genes at a time. These modifications
include the following gross structural
rearrangements.
 Deletion: loss of genes
 Inversion: alteration in gene order
 Duplication: duplication of genes
 Translocation: segments of chromosomes
moving to new locations.





Epigenetic changes somaclonal variation can be
temporary and over time are reversible.
However, sometimes they can persist through
the life of the regenerated plant.
One common phenotypic change seen in plants
produced through tissue culture is rejuvenation.
Rejuvenation causes changes in morphology such
as earlier flowering and enhanced adventitious
root formation.
Epigenetic changes may be caused by DNA
methylation and thus may be one of the
important causes of somaclonal variation.





As plant tissues are composed of heterogeneous
array of cells of various ages, different
physiological states and degree of differentiation
and cells with different ploidy level exist.
By placing cells in tissue culture, the genome at
different molecular states is suddenly placed
under stress to cope with in vitro conditions.
It has also been reported that changes in tissue
culture conditions could influenced the
frequency of variation.
The end effect seems to be an array of genetic
engineering changes.
Studies concerning different aspects of
somaclonal variation are important for the
following reasons.
 Firstly, however successful utilization of SC variation heavily depends
upon its systematic evaluation and judicious utilization in breeding
programmes. This demands appropriate experimentation.
 Secondly, SC variation is of interest as a basic genetic process, since it
contradicts the concept of clonal uniformity. The cells and tissues
which are expected to produce true to type plants through the
processes of de-differentiation, division and re-differentiation, possibly
perceive the whole process as stress, as a result of which the genome,
known for its plasticity, restructures itself to modulate the expression
of gene as demanded by the in vitro conditions.
 Third , SC variation is unwanted when the objective is
mricropropagation of elite genotypes or genetic transformation that
partly involved tissue culture. Under such circumstances, prevention or
at least minimization of variation is of utmost importance. To achieve
this, the frequency, nature and magnitude of somaclonal variation in
relation to manipulation of media components, explant source, culture
conditions etc. should essentially be understood.
 Majority of studies undertaken on SC variation are confined
to early generation of soma clones.
 Therefore information on the nature, inheritance pattern and
stability of morphological and molecular changes expressed
in the advanced generation of soma clones is lacking.
 The different aspects of somaclonal variation investigated so
far are as follows
1) Generation of variation
2) Characterization of variant for morphological traits
3) Analysis of biochemical and chromosomal basis of variation
4) Relating the variation to alteration in DNA
The mechanism of somaclonal variation
According to Bhaskaran (1985) variations in somaclones occur due to the following
reasons:
1.
The pre-existing genetic variations in the explant tissue,
2.
The spontaneous mutations that can accumulate during the many division cycles
that cell of the explant go through before differentiating into an in vitro plant. The
recessive mutation will naturally require a method by which they can express
even diploid cells. Somatic crossing over followed by segregation is a likely
mechanism, for the homozygosity and thus phenotypic expression of the recessive
(Chopra and Sharma, 1988).
3.
Intracellular mutagenic agents produced during in vitro growth.
4.
Numerical and structural changed in chromosomes during in vitro growth.
5.
Activation of transposable elements or jumping genes, are genetic entities which
have the locus at which they get integrated is matured.
6.
Agriculturists are very hopeful about practical advantages of somaclonal variation
and they are waiting when this technique is fully integrated with the conventional
plant breeding procedures.






Take an aliquot of suspension and filter off the culture through a
wire mesh (300mm). note the volume of the filtrate (F) containing
single cells and small clumps and place the drop of this
suspension to heamocytometer to determine the number of cells
by the equation
N = P x 100 x F 0.1 mm
where, N = total number of cells and clumps, P = number of
cells in the squares of the haemocytometer, f = volume of the
filtrate.
Forms of somaclonal variation:
Many different forms of somaclonal variation arise. The most
common forms include point mutation, chromosomal aberrations
and increase or decrease in the number of nuclear
chromosomes.
It is important to realize that not all forms of variability that arise
in vitro are heritable. Some morphological and biochemical
variants are due to physiological effects and are not exhibited in
subsequent generations.





There are several different approaches to detecting and
isolating somaclaonal variants from cultured plant cell
populations.
Morphologically distinct cells such as nonphotosynthetic
(nongreen) cells or cells that accumulate anthocyanin and
other plant pigments are detected visually.
To isolate herbicide- and antibiotic-resistant variants,
plant cells are simply grown on media containing of the
wild type cells in a culture.
The surviving cells are then subcultured and retested for
growth on herbicide or antibiotic supplemented medium.
Through this method, one can eliminate any remaining
wild-type cells that may have inadvertently survived the
first round of selection.
Applications in plant breeding
•
•
•
•
•
•
•
Somaclonal variation and gemetoclonal variation are the important source of
introducing genetic variation that could be of value to plant breeders.
Single gene mutation in the nuclear or organelle genome usually provides the best
available variety in vitro which has a specific improved character.
Somaclonal variations are used to uncover new variant retaining all the favorable
characters along with an additional useful trait, e.g., resistance to disease or an
herbicide.
These variants can then be field tested to ascertain their genetic stability.
Gametoclonal variation is induced by meiotic recombination during the sexual
cycle of the F1 hybrid results in transgressive segregation to uncover unique gene
combinations.
Various cell lines selected un vitro and plant regenerated through it prove
potentially applicable to agriculture and industry specially resistance to herbicide,
pathotoxin, salt or aluminium, useful in the synthesis of secondary metabolites on
a commercial scale, etc.
The techniques used for development of somaclonal and gametoclonal variation
are relatively easier than recombinant DNA technology and is the appropriate
technology for genetic manipulation of some crops.






The main factors that influence the variation
generated from tissue culture are
(1) the degree of departure from organised
growth,
(2) the genotype,
(3) growth regulators and
(4) tissue source.
Despite an increasing understanding of how
these factors work it is still not possible to
predict the outcome of a somaclonal
breeding programme.








New varieties have been produced by somaclonal variation,
but in a large number of cases improved variants have not
been selected because
(1) the variation was all negative,
(2) positive changes were also altered in negative ways,
(3) the changes were not novel, or
(4) the changes were not stable after selfing or crossing.
Somaclonal variation is cheaper than other methods of
genetic manipulation.
At the present time, it is also more universally applicable
and does not require ‘containment’ procedures.
It has been most successful in crops with limited genetic
systems and/or narrow genetic bases, where it can provide
a rapid source of variability for crop improvement.