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SOMATIC EMBRYOGENESIS

AND

ORGANOGENESIS

BACKGROUND AND DISCUSSIONS


ORGANOGENESIS
In organogenesis, cultured explants (e.g cotyledons,
hypocotyls, stem, leaf, shoot apex, root, flowers, petioles and
embryos) are induced to develop adventitious roots, shoots and
other organs.

Organogenesis can be induced in specific nutrient media


containing balanced ratio of plant growth regulators such as
auxin and cytokinin.

In such cases, plant regeneration from cultured cells may occur


through shoot-bud differentiation. The cells of leaf cultures of
some species are able to directly differentiate shoots and roots,
leading to the production of plantlets.
ORGANOGENESIS
There are three methods of plant regeneration via
organogenesis:

5. Depends on adventitious organs arising from a callus culture


or alternatively,

7. Directly from an explant.

9. Axillary bud formation and growth from some explants.


FACTORS AFFECTING ORGANOGENESIS
Organ formation is controlled by quantitative interaction (ratio
rather then absolute concentration) of constituents in growth and
development medium.

Generally, cytokinin (e.g. adenine or kinetin) in the medium leads


to the production of shoot differentiation and development.
Reports indicate that kinetin is 30,000 times more potent than
adenine. The shoot-forming effect is modified by auxin (IAA and
NAA), which at lower concentration (5 µM IAA) suppresses the
differentiation of shoot in tobacco callus.

A relatively high concentration of auxin seem to favour cell


proliferation and root differentiation, while higher levels of
cytokinin promote shoot differentiation.
FACTORS AFFECTING ORGANOGENESIS
The inhibitory effect of auxin can be counteracted by increased
levels of phosphate ions in the medium and this promotes shoot
formation in the absence of cytokinin.

Casein hydrolysate or tyrosine also induces kinetin type shoot


formation even in the presence of higher levels of IAA in the
medium.

The requirement for exogenous auxin and cytokinin in the


process of shoot differentiation varies with the tissue system and
apparently depends on the endogenous levels of the two
hormones in the tissue, viz a viz. an auxin and cytokinin.

Endosperm cultures require cytokinin alone or in combination


with a very level of auxin for shoot differentiation.
FACTORS AFFECTING ORGANOGENESIS
Polyamines have been shown to be associated with the
induction of cell division, growth and differentiation of plant cells.

Other cytokinins which influence the induction of shoots include


BA, 2-iP and Zeatin.
FACTORS AFFECTING ORGANOGENESIS
In alfalfa and cereals, a two step process of organogenic
differentiation occurs. Alfalfa callus initiated on a medium
(induction medium) containing higher auxin (2,4-D) to kinetin,
favours shoot-formation. Whereas, a higher proportion of kinetin
to 2,4-D in the medium (regeneration medium) supports root
induction.

The cereal callus, on the other hand, is initiated in the induction


medium containing 2,4-D and kinetin but organogenesis occurs
only when pieces of callus are transferred to a hormone-free
regeneration medium
PHYSICAL FACTORS AFFECTING ORGANOGENESIS

Light Intensity

In the case of Pelargonium callus maintained under continuous


light or high light intensity remains whitish and does not exhibit
organogensis. Such light intensity has also been shown to be
inhibitory for shoot formation in tobacco.

The quality of light also influence organogenic differentiation.


Blue light promotes shoot differentiation in tobacco calus while
red light stimulates rooting.

In general, maintenance of callus under alternating light and


dark periods (15-16h) may prove satisfactory for differentiation
of shoots.
PHYSICAL FACTORS AFFECTING ORGANOGENESIS

Temperature

Affects the callus growth and differentiation. Increases in


temperature up to 33ºC may be associated with the rise in the
growth of tobacco callus but for differentiation of shoot, a lower
temperature (18ºC) may be optimal.

Physical state of medium

Medium solidified with agar favour shoot formation.

Physiology of explant

Genome and the physiological state of the explant are other


factors accounting for differentiation in culture.
Somatic Embryogenesis (SE)
SE is the process of a single plant cell or a group of callus
initiating the developmental pathway that leads to reproducible
regeneration of a non-zygotic embryo capable of germinating to
form complete plants.

SE occurs most frequently in tissue culture and as an


alternative ORGANOGENESIS for regeneration of whole plant.

Adherence to SE (this pattern of morphogenesis) depends on


the co-ordinated behaviour of a cell or cells to establish polarity
as a unit and thereby initiate gene action sequentially specific to
emerging tissue regions.
Somatic Embryogenesis
SE is initiated by ‘pre-embryogenic determined cells’ (PEDC) or
by ‘induced embryogenic determined cells’ (IEDC).

In PEDCs the embryogenic pathway is predetermined and the


cells appear to only wait for the synthesis of an inducer (or
removal of an inhibitor) to resume independent mitotic divisions
to express their potential.

Such cells are formed in embryogenic tissues (young tissues)


of in vitro plants e.g. embryo-sac tissues within ovules.

In SE, embryo-like structures, which can develop into whole


plants, are formed from somatic tissues.
Somatic Embryogenesis

IEDCs require redetermination to the embryogenic state by


exposure to specific growth regulators (2,4-D). These cells are
differentiated generally in microspore callus cultures.

Once the embryogenic state has been reached, both cell type
proliferate in a similar manner as embryogenic determined cells
(EDCs). Plantlets are then produced directly by following the full
embryogenic pathway as a co-ordinated group of EDCs.

Somatic embryos may develop form single cells or form a small


group of cells.

Repeated cell divisions lead to the production of a group of


cells that develop into organised structure known as a ‘globular-
stage embryo’.
Somatic Embryogenesis

Further development results in heart- and torpedo-stage


embryos, from which plants can be regenerated.

Polarity is established early in embryoid development. Signs of


tissue differentiation become apparent at the globular stage and
apical meristems are appparent in heart-stage or cotyledon-
stage embryoids.
Somatic Embryogenesis
SE can be of direct and indirect types.

In direct, SE, the embryoids are formed directly form a cell or


small group of cells without the production of an intervening
callus (common among reproductive tissues). It is rare in
comparison with indirect SE.

e.g. Young leaf explants from alfalfa (Medicago falcata), washed


in plant growth regulator-free medium and placed in liquid
medium (B5) supplemented with 2,4-D (4 mgl-1), kinetin (0.2 mgl-
1
), adenine (1 mgl-1) and glutathione (10 mg-1). The cultures are
maintained in agitated liquid medium for about 10-15 days.
Washing the explants and replacing the old medium with B5
medium supplemented with maltose and polyethylene glycol
results in the development of somatic embryoids. These somatic
embryoids can be matured on solid medium containing abscisic
acid.
Somatic Embryogenesis
In Indirect SE, callus is first produced from the explant.

Embryoids can then be produced from the callus tissue or from


cell suspension produced from the callus.

e.g. Callus can be established from explants from a wide range


of carrot tissues by placing the explant on solid medium (M&S)
containing 2,4-D (1 mgl-1). This callus can be used to produce
cell suspension by placing it in agitated liquid MS meduim
containing 2,4-D (1 mg-1). Replacing old medium and replacing
with fresh medium containing abscisic acid (0.025 mgl-1) results
in the production of embryoids.
SE IN CALLUS CULTURES
SE is achieved in two steps:

V. Callus is initiated, multiplied on a medium rich in auxin


(Proliferation) Medium-PM, e.g. with 2,4-D) which induces
differentiation of localised groups of the meristematic cells
‘embryogenic clumps’ (ECs).

• The ECs then develop into mature embryoids when


transferred to a medium with a very low level of auxin or no
auxin at all (Embryoid Development Medium-EDM).

Thus embyogenic callus consist of proembryoids and these


embryo-like structures are normally bipolar units and may
(capable) germinate into full plantlets under suitable culture
conditions.

Non-zygotic embryos are somatic embryoids or


OTHER FACTORS ASSOCIATED IN SE
Nitrogen source

A substantial amount of nitrogen, usually in reduced form


such as ammonium salts, is required for both embryo
initiation and maturation.

However, a combination of reduced nitrogen and nitrate in


the medium appears beneficial for a number of culture
systems.

The source of nitrogen can be in the form of complex


addenda (coconut milk, casein hydrolysate, a mixture of
amino acids and ammonium ions).
OTHER FACTORS ASSOCIATED IN SE
Media Components

Half normal concentration of media components and in some


cases lacking phytohormones.

Light

Light intensities, particular wavelengths and photoperiods,


moderate and stimulate the formation and development of
embryoids.

Exudates

Extracts (tomato, banana, yeast / coconut water)


OTHER FACTORS ASSOCIATED IN S.E.
Other Constituents

The amount of dissolved oxygen (DO). The effect of carbon


source can influence the osmotic value.

Activated charcoal is reported to increase embryogenesis. As


certain tissues release volatile substances which can inhibit
SE in the callus.
APPLICATION OF SE
Clonal propagation (large-scale propagation of plantlets)

Raising somoclonal variants (new trait of plant-new variety)

Cloning zygotic embryos for repetitive SE

Synthesis of artificial seeds (by encapsulation of somatic


embryos)

Genetic transformation or plant improvement via SE


FLOW SHEET SUMMARISING THE INTER-RELATIOSHIPS
BETWEEN THE DIFFERENT LINES OF
EXPERIMENTATION OPENED UP BY THE TECHNIQUES
OF PLANT TISSUE AND CELL CULTURE