INTRODUCTION
The fertilization process triggers the egg cell
(called the zygote after fertilization) to divide and develop into an embryo
(the process of embryo development is called embryogenesis). However, fertilization
is not always essential to stimulate the egg to undergo embryogenesis. As happens
in parthenogenesis, the pollination stimulus alone, or simply the application
of some growth regulators may induce the egg to undergo embryogenic
development. Moreover, it is not the monopoly of the egg to form an embryo. Any
cell of the female gametophyte (embryo sac), or even that of the sporophytic
tissue around the embryo sac may give rise to an embryo. In several species of
Citrus and Mangifera the development of adventive embryos from nucellar cells
is a normal feature.
However, the nucellar embryos attain maturity only if
they are pushed into the embryosac at an early stage of development, or else
they fail to mature. In nature there is no instance of ex-ovulo embryo
development (Bhojwani and Bhatnagar, 1990). These in vivo observations would
suggest that for their growth and development embryos require a special
physical and chemical environment available only inside the 'magic bath' of the
embryo sac.
During the last three decades considerable information
has accumulated to establish the embryogenic potential of somatic plant cells,
and there has been an explosion in the number of species that form somatic embryos
(SEs).
Based on the recent spectacular development in cell
and tissue culture of higher plants it would be fair to say that any cell, in which
irreversible differentiation has not proceeded too far, will, if placed in an
appropriate medium, develop in an embryo-like way and produce a complete plant.
The whole complex sexual apparatus is, therefore, not an essential prerequisite
for cells to acquire embryonic properties. The events occurring in the ovule
after fertilization thus provide only a specia1 case of embryogeny.
An embryo derived from a somatic cell, other than zygote, on a culture medium invitro is somatic embryo and the process is known as somatic embryogenesis.
The process in which a single or a group of somatic cells initiate the developmental pathway that leads to the formation of non-zygotic embryos is called Somatic Embryogenesis. (Zygotic embryos develop from fertilized eggs termed zygote whereas non-zygotic embryos develop from cells other than zygote).
Based on the induction of somatic embryogenesis, it is
of two types:
Direct Somatic Embryogenesis:
The development of somatic embryos directly on the
explant without undergoing callus formation is referred to as direct somatic
embryogenesis. This is possible due to the presence of pre-embryonic determined
cells (PEDC) found in certain tissues of plants.
Indirect Somatic Embryogenesis:
Induction of somatic embryogenesis through callus is
called as indirect somatic embryogenesis. Indirect somatic embryogenesis is
commercially very attractive since a large number of embryos can be generated
in small volume of culture medium. The somatic embryos so formed are
synchronous and with good regeneration capability.
In somatic embryogenesis (SE),
embryo-like structures analogous to zygotic embryo are formed either directly
from the tissue or via an intervening callus phase. The process is opposite of
zygotic or sexual
In either of
the cases, the somatic embryos resemble the zygotic embryos. In dicotyledonous
plants, the somatic embryos passes through the globular, heart, torpedo and
cotyledonary stages, as happens in zygotic embryos. The embryos germinate and
develop into complete plantlets. The only major difference between somatic and
zygotic embryogenesis is that somatic embryos do not pass through the
desiccation and dormancy phases as happens in zygotic embryos, but rather
continue to participate in the germination process.
Whether originating directly or indirectly via callusing, somatic embryos arise from single special cells located either within clusters of meristematic cells in callus mass or in the explant tissue. Somatic embryogenesis is regarded as a three step process:
- i. Induction of embryo
ii. Embryo development
iii. Embryo maturation
Protocol for
Inducing Somatic Embryogenesis
The plant
material Daucus carota represents the classical example of SE in culture
1.
Leaf Petiole (0.5 – 1 cm) or root segments from
the seven day old seedlings (1 cm) or cambium tissue ( 0.5 cm) from storage
root can be used as explants.
2.
Following aseptic technique, explants are placed
individually on a semi-solid MS 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 transforming 0.2gm of callus tissue to 250ml of
Erlenmeyer flask containing 20-25 ml of liquid medium of the same composition
as used for callus growth. Flasks are placed on a horizontal gyratory shaker
with 125 – 160 rpm at 250C.
4.
Cell suspensions are sub cultured every 4 weeks
by transferring 5 ml to 65 ml fresh liquid medium.
5.
To induce a more uniform embryo population, cell
suspension is passed through a series of stainless steel mesh sieves. To induce
SE, portions of sieved cells suspensions are transferred to 2,4,-D free liquid
medium or cell suspension can be placed in semi-solid MS medium devoid of
2,4-D.
Form normal embryo development and to inhibit precocious germination
especially root elongation, 0.1 – 1 µM ABA can be added.
6.
After 3-4 weeks, the culture would contain
numerous embryos in different stages of development.
Applications
of somatic embryogenesis
Following
features of somatic embryos prompted many scientists to achieve regeneration
via somatic embryogenesis using various explants, most popular ones being
zygotic embryos, or excised cotyledons or hypocotyls
i. The embryo culture technique is applied to overcome embryo
abortion, seed dormancy and self-sterility in plants.
- ii. Somatic embryogenesis offers immense
potential to speed up the clonal propagation of plants being bipolar in
nature.
iii. Being single cell in origin, there is a possibility to
automate large scale production of embryos in bioreactors and their field
planting as synthetic seeds.
iv. The bipolar nature of embryos allows their direct development
into complete plantlet without the need of a rooting stage as required for
plant regeneration via organogenesis.
v. Epidermal single cell origins of embryos favor the use of this
process for plant transformation.
vi. It can also be used for the production of metabolites in
species where embryos are the reservoir of important biochemical compounds.
vii. The production of artificial seeds using somatic embryos is an
obvious choice for efficient transport and storage.
Synthetic
Seeds or Artificial Seeds
Seed is the structure that develops from and
ovule after fertilization and contains the embryonic plant with food reserves.
Artificial
seeds are living seeds-like structure derived from somatic embryoids invitro
culture after encapsulation by a hydrogel. The preserved embryoids are termed artificial
seeds.
The encapsulated embryoids can resist
unfavourable conditions without desiccation. They can be shown directly in the greenhouse
or in fields.
Kitto and Janic produced the first synthetic
seed from carrot developed embryoids. Polyoxyethylene was used as protective
coat for synthetic seeds.
Schematic representation of steps of Synthetic seeds
production:
Establishment of callus culture
Induction of somatic embryogenesis
Maturation of somatic embryos
Encapsulation of somatic embryos
Evaluation of embryoid and plant conversion
Two types of synthetic seeds are produced:
I. Desiccated synthetic seeds II. Hydrated synthetic
seeds
I. Desiccated synthetic seeds : It involves encapsulation
of somatic embryos followed by their desiccation and can be prepared by
following methodology:
The polyox is
readily soluble in water and dries to thin film. It does not support the growth
of microorganism and is non toxic to the embryos. Embryo survival and
conversion of seeds are determined by redissolving the wafers in embryogenic
medium and culturing the rehydrated embryos.
II. Hydrated synthetic seed: Several methods have
been examined to produce hydrated artificial seeds of which Ca-alginate
encapsulation has been the most widely used. It can be prepared by following
steps:
isolated
somatic embryos are mixed with 0.5 to 5% (W/V) Sodium alginate and dropped
into 30-100 µM Calcium nitrate solution. Surface complexation begins
immediately and the drops are gelled completely within 30 minutes
Uses of Synthetic
seeds:
1.
The seeds are formed in only one season of the
year in natural conditions, but synthetic seeds can be produced throughout the
year.
2.
Natural seeds undergo seed dormancy. This dormancy
period is reduced in synthetic seeds. Plants take less time to grow.
3.
Germplasm storage is easy.
4.
Hybirds, Genetically modified plants/crops can
be easily propagated with synthetic seeds
5.
Genetic uniformity is maintained in synthetic
seeds.
6.
Artificial seeds can be employed for production
of polyploids with elite traits, avoiding the genetic recombination when these
plants are propagated using conventional plant breeding systems, thus saving on
time and costs
7.
Artificial seeds can be also used in the
proliferation of male or female sterile plants for hybrid seed production
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