Embryogenesis?

In plant tissue culture, the developmental pathway of numerous well-organised, small

embryoids resembling the zygotic embryos from the embryo genic potential somatic plant
cell of the callus tissue or cells of suspension culture is known as somatic embryogenesis.

What is Embryo genic Potential?

The capability of the somatic plant cell of a culture to produce embryoids is known as
embryo genic potential.

What is Embryoid?
Embryoid is a small, well-organised structure comparable to the sexual embryo, which is
produced in tissue culture of dividing embryo genic potential somatic cells.

Principles of Somatic Embryogenesis:

Somatic embryogenesis may be initiated in two different ways:
1. In some cultures somatic embryogenesis occurs directly in absence of any callus pro-
duction from “pro-embryo genic determined cells” that are already programmed for embryo
differentiation (Fig 8.1). For instance, somatic embryos has been developed directly from
leaf mesophyll cells of orchard grass (Dactyhs glomerata L.) without an intervening callus
tissue.
Explants, made from the basal portions of two innermost leaves of orchard grass were
cultured on a Schenk and Hildebrandt medium supplemented with 30 µM 3, 6-dichloro-O-
anisic acid (dicamba). Plant formation occurred after sub culturing the embryos on the
same medium without dicamba (Conger et al., 1983).
2. The second type of somatic embryo development needs some prior callus formation and
embryoids originate from “induced embryo genic cells” within the callus tissue.
In most of the cases, indirect embryogenesis occurs. For indirect somatic embryogenesis
where it has been induced under in vitro condition, two distinctly different types of media
may be required—One medium for the initiation, of the embryonic cells and another for the
subsequent development of these cells into embryoids.

The first or induction medium must contain auxin in case of carrot tissue and somatic
embryogenesis can be initiated in the second medium by removing the hormone or lowering
its concentration. With some plants, however, both embryo initiation and subsequent
maturation and subsequent maturation occur on the first medium and a second medium is
required for plantlet development.

In some cases, a given culture may differentiate the embryo genic cells, but their further
growth may be blocked by an imbalance of nutrition in the culture medium. According to
Kohlenback, (1978), abnormalities known as embryonal budding and embryo genic clump
formation may occur, if relatively high level of auxin is present after the embryo genic cells
have been differentiated.

Embryoids are generally initiated in callus tissue from the superficial clumps of cells (pri-
mordia) associated with enlarged vacuolated cells that do not take part in embryogenesis.
The embryo genic cells are generally characterised by dense cytoplasmic contents, large
starch grains, a relatively large nucleus with a darkly stained nucleolus. In suspension
culture, embryoids do not form suspended single cell, but form cells lying at or near the
surface of the small cell aggregates.

Each developing embryoid of carrot passes through three sequential stages of embryo for-
mation such as globular stage, heart-shape stage and torpedo stage (Fig 8.3). The torpedo
stage is a bipolar structure which ultimately gives rise to complete plantlet. The culture of
other plants may not follow such sequential stages of embryo development.
In general, somatic embryogenesis occurs in short-term culture and this ability decreases
with increasing duration of culture. But there are some exceptional cultures where
embryogenesis has been reported from the callus tissue maintained over a period of year.
According to Smith and Street, (1974), changes in ploidy of the cultured cell may lead to loss
of embryo genic potential in long term culture. The loss of embryo genic potential in long
term culture may also result from loss of certain biochemical properties of the cell.

In callus culture or in suspension culture, embryoid formation occurs asynchronously. Some

progress has been made in inducing synchronization of somatic embryogenesis in cell
suspension culture. A high degree of synchronization has been achieved in a carrot
suspension culture by sieving the initial cell population.

Somatic Embryogenesis in Culture:

The plant material Daucus carota represents the classical example of somatic embryo-
genesis in culture.

The protocol is described below:

1. Leaf petiole (0.5-1 cm) or root segments from seven-day old seedlings (1 cm) or cambium
tissue (0.5 cm3) from storage root can be used as explant. Leaf petiole and root segment can
be obtained from aseptically grown seedlings (Cambium tissue can be obtained from surface
sterilized storage tap root 2. Following aseptic technique, explants are placed individually
on a semi-solid Murashige and Skoog’s medium containing 0.1 mg/L 2, 4-D and 2% sucrose.
Cultures are incubated in the dark. In this medium the explant will produce sufficient callus
tissue.
3. After 4 weeks of callus growth, cell suspension culture is to be initiated by transferring
0.2 gm. of callus tissue to a 250 ml of Erlenmeyer flask containing 20-25 ml of liquid
medium of the same composition as used for callus growth (without agar). Flasks are placed
on a horizontal gyratory shaker with 125-160 rpm at 25°C. The presence or absence of light
is not critical at this stage.

4. Cell suspensions are sub-cultured every 4 weeks by transferring 5 ml to 65 ml of fresh

liquid medium.

5. To induce a more uniform embryo population, cell suspension is passed through a series
of stainless steel mesh sieves. For carrot, the 74 µ sieve produces a fairly dense suspension
of single cell and small multiple clumps. To induce somatic embryogenesis, portions of
sieved cell suspension are transferred to 2, 4-D free liquid medium or cell suspension can be
planted in semi-solid MS medium devoid of 2, 4-D. For normal embryo development and to
inhibit precocious germination especially root elongation, 0.1- 1 µM ABA can be added to
the culture medium. Cultures are incubated in dark.

6. After 3-4 weeks, the culture would contain numerous embryos in different stages of de-
velopment.

7. Somatic embryos can be placed on agar medium devoid of 2, 4-D for plantlet develop-
ment.

8. Plantlets are finally transferred to Jiffy pots or vermiculite for subsequent development.

1) Chemical Factors:
a. Auxin:
Somatic embryogenesis in carrot is a classic example. It is a two-step process. The carrot
cells first develop into a callus tissue in the medium containing the auxin, namely 2, 4-D
(0.5-1 mg/L).

When such callus tissue is transferred to the same medium with a very low level of auxin or
no auxin at all, embryoids are formed. If the callus tissue is maintained continuously in the
medium containing 2, 4-D, embryoids would not form.

Similarly, if the carrot cells are maintained continuously from the initial step in auxin-free
medium, embryoids do not develop. Therefore, the presence of auxin in the first step is
possibly essential for the proliferation of callus tissue and for the induction of embryo genic
potential cells.

In the second step, auxin is no longer required for the embryo genic potential cells to form
embryoids. Like carrot, two-step process of in vitro development of somatic embryo is also
found in Coffea arabica. Other than 2, 4- D, naphthalene acetic acid (NAA), indole butyric
acid (IB A) have also been used in other culture system for the induction of embryo genic
potential cells.
In Citrus sinensis, the callus tissue is initiated from the nucellar tissue in the medium
containing IAA and Kinetin. Such medium is required for the callus growth and embryo dif-
ferentiation. After repeated subculture in the same medium, the callus tissue shows a
gradual decline in somatic embryogenesis.

On the other hand, when such callus tissue is transferred to auxin-free medium, it again
improves the callus growth and embryogenesis. At that time even the addition of very low
concentration of IAA inhibits the process of somatic embryogenesis. Therefore, it appears
that after a prolonged period of culture, the callus tissue may become habituated or
phytohormone autonomous.

This means that they are now able to grow on a standard medium which is devoid of growth
hormones. The cells appear to have developed the capacity to synthesise adequate amount
of both auxin and cytokinin which they required for the growth and somatic embryogenesis.
But after few subcultures, the habituated culture of C sinensis again shows a decline in the
embryo genic potential.

When the habituated callus tissue is exposed to irradiation, the process of somatic
embryogenesis is again improved. Irradiation is known to breakdown auxin. So this ob-
servation reveals that high level of endogenous auxin produced by habituated callus tissue
inhibits the process of somatic embryogenesis. But when the tissue is irradiated, the high
level of endogenous auxin is lowered and a minimal level of auxin is responsible for the
process of somatic embryogenesis.

Thus, from the above experimental evidences, it appears that a minimal level of auxin is
essential for the induction of embryo genic potential cells within the cultured tissue but for
the organization and maturation of the embryoids from the embryo genic potential cell,
auxin does not play any positive role.

b. Cytokinin:
The effect of cytokinins in embryogenesis is somewhat obscure because of conflicting re-
sults. In carrot suspension culture, zeatin (0.1 µM) a type of cytokinin, stimulates embryoge-
nesis when the cells are sub-cultured in auxinfree medium. But the process is inhibited by
the addition of either kinetin or benzylaminopurine (BAP) to the medium.

The inhibitory effect of cytokinins may be due to selective stimulation of cell division of non-
embryo genic cells of the culture. Stimulatory effect of cytokinin has also been reported in
some specific culture system Stewart et al. (1964) also reported the importance of coconut
milk (containing a source of cytokinin) for somatic embryogenesis.

c. Gibberellin:
Gibberellin has no positive effect. In carrot and Citrus, gibberellin inhibits somatic embryo-
genesis.

d. Reduced Nitrogen:
Substantial amount reduced nitrogen (NH4) are required for embryogenesis. In carrot
culture, the addition of NH4Cl to the embryo genie medium already containing KNO3produ-
ces near-optimal numbers of embryoids. It is, therefore, convenient to use NH+4 in combina-
tion with NO–3. But no other form of inorganic reduced nitrogen has been as effective as
NH+4 for somatic embryogenesis.
Glutamine, glutamic acid, urea and alanine are found to partially replace NH4Cl as a supple-
ment to KNO3. These various nitrogen sources are not specific for the induction of
embryogenesis, although, at low concentration organic forms are much more effective than
inorganic nitrogen compounds.
(2) Other Factors:
The medium supplemented with activated charcoal has facilitated embryogenesis in several
culture. The induction of embryogenesis is achieved successfully by the addition of charcoal
when auxin depletion in the medium fails to produce the desired results.

It has been suggested that charcoal may absorb a wide variety of inhibitory substances as
well as hormone. Optimal level of dissolved oxygen and high potassium in the medium are
necessary for embryogenesis. But in Citrus, certain volatile and non-volatile substances
inhibit embryogenesis.