4.

Somatic Embryogenesis:
The process of regeneration of embryos from somatic
cells, tissues or organs is regarded as somatic (or asexual)
embryogenesis. Somatic embryogenesis may result in
non-zygotic embryos or somatic embryos (directly formed
from somatic organs), parthenogenetic embryos (formed
from unfertilized egg) and androgenic embryos (formed
from male gametophyte).
In a general usage, when the term somatic embryo is used
it implies that it is formed from somatic tissues under in
vitro conditions. Somatic embryos are structurally similar
to zygotic (sexually formed) embryos, and they can be
excised from the parent tissues and induced to germinate
in tissue culture media.
Development of somatic embryos can be done in plant
cultures using somatic cells, particularly epidermis,
parenchymatous cells of petioles or secondary root
phloem. Somatic embryos arise from single cells located
within the clusters of meristematic cells in the callus or
cell suspension. First a pro-embryo is formed which then
develops into an embryo, and finally a plant.
Two routes of somatic embryogenesis are known — direct
and indirect (Fig 47.6).
Direct Somatic Embryogenesis:
When the somatic embryos develop directly on the excised plant (explant) without undergoing callus formation, it is
referred to as direct somatic embryogenesis (Fig 47.6A). This is possible due to the presence of pre-embryonic
determined cells (PEDC) found in certain tissues of plants. The characteristic features of direct somatic embryogenesis
is avoiding the possibility of introducing somaclonal variations in the propagated plants.
Indirect Somatic Embryogenesis:
In indirect embryogenesis, the cells from explant (excised plant tissues) are made to proliferate and form callus, from
which cell suspension cultures can be raised. Certain cells referred to as induced embryo genic determined cells (IEDC)
from the cell suspension can form somatic embryos. Embryogenesis is made possible by the presence of growth
regulators (in appropriate concentration) and under suitable environmental conditions.
Somatic embryogenesis (direct or indirect) can be carried on a wide range of media (e.g. MS, White’s). The addition of
the amino acid L-glutamine promotes embryogenesis. The presence of auxin such as 2, 4-dichlorophenoxy acetic acid
is essential for embryo initiation. On a low auxin or no auxin medium, the embryo genic clumps develop into mature
embryos.
Indirect somatic embryogenesis is commercially very attractive since a large number of embryos can be generated in a
small volume of culture medium. The somatic embryos so formed are synchronous and with good regeneration
capability.
Artificial Seeds from Somatic Embryos:
Artificial seeds can be made by encapsulation of somatic embryos. The embryos, coated with sodium alginate and
nutrient solution, are dipped in calcium chloride solution. The calcium ions induce rapid cross-linking of sodium
alginate to produce small gel beads, each containing an encapsulated embryo. These artificial seeds (encapsulated
embryos) can be maintained in a viable state till they are planted.
Somatic 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?

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.
Brief Historical Background:
J. Reinert (1958-59):
Reported his first observations of in vitro somatic embryogenesis in Daucus carota.
F. C. Steward, M. O. Mapes and K. Mears (1958):
:
Also reported the somatic em­bryogenesis in carrot from freely suspended cells and emphasized the importance
of coconut milk for in vitro somatic embryogenesis.
N. S. Rangaswamy (1961):
Studied in detail the somatic embryogenesis in Citrus sp.
R. N. Konar and K. Nataraja (1969):
Studied the somatic embryogenesis of Ranuncu­lus sceleratus using various floral parts (including anthers) as
well as somatic tissues in culture.
P. V. Ammirato (1974):
Reported the effect of abscisic acid on the development of so­matic embryos from cells of Carum carvi.
H. Lang and H. W. Kohlenbach (1978):
Demonstrated the ability of mechanically iso­lated, fully differentiated mesophyll cells of Macleaya cordata to
yield an embryogenic cal­lus.:
B. V. Conger, G. E. Hanning, D. J. Gray and J. K. McDaniel (1983):
Obtained direct embryogenesis from leaf mesophyll cells of orchard grass (Dactyhs glomerata L.) without an
intervening callus tissue.
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 pro­duction 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.
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
occur on the first medium and a second medium is re­quired for plantlet development.
Embryoids are generally initiated in callus tissue from the superficial clumps
of cells (pri­mordia) 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 (Fig 8.2).

Each developing embryoid of carrot passes through three sequential stages

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 develop­ment.

8. Plantlets are finally transferred to Jiffy pots or vermiculite for subsequent development.
Importance of Somatic Embryogenesis:
The potential applications and importance of in vitro somatic embryogenesis and organo­genesis are
more or less similar. The mass pro­duction of adventitious embryos in cell culture is still regarded by
many as the ideal propagation system. The adventitious embryo is a bipolar structure that develops
directly into a complete plantlet and there is no need for a separate root­ing phase as with shoot
culture.

Somatic em­bryo has no food reserves, but suitable nutrients could be packaged by coating or
encapsulation to form some kind of artificial seeds. Such ar­tificial seeds produce the plantlets directly
into the field. Unlike organogenesis, somatic embryos may arise from single cells and so it is of
special significance in mutagenic studies.

Plants derived from asexual embryos may in some cases be free of viral and other pathogens. For an
example, Citrus plant propagation from embryo genic callus of nuclear origin are free of Virus. So it is
an alternative approach for the production of disease-free plants.