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Embryogenesis -Somatic embryogenesis is the process of a single cell or group of cells initiating the developmental pathway that leads to reproducible regeneration of nonzygotic embryos capable of germinating to form complete plants.
Under natural conditions this pathway is not normally followed but from tissue cultures somatic embryogenesis occurs more often and as an alternative to organogenesis for regeneration of whole plants.
According to Sharp et al. (1982), somatic embryogenesis is, initiated either by preembryogenic determined cells or by induced embryogenic determined cells.
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In preembryogenic determined cells, the embryogenic pathway is pre determined and the cells appear to only wait for the synthesis of an inducer (or removal of an inhibitor) to resume independent mitotic divisions in order to express their potential. Such cells are found in embryonic tissues (including scutellum of cereals), certain tissues of young in vitro grown plantlets, the nucellus, and the embryo sac (within ovules of mature plants).
Induced embryogenic determined cells, on the other hand, require redetermination to the embryogenic state by exposure to specific growth regulators such as 2, 4-D. These cells are dedifferentiated generally in microspore (anther) cultures and callus cultures. Once the embryogenic state has been reached both cell types proliferate in a similar manner as embryogenic determined cells.
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Plant lets are then directly produced by following the full embryogenic pathway as a coordinated group of embryogenic determined cells. Some times individual cell or cells from the group may escape and give rise to either embryoids or nodular embryogenic callus (e.g. scutellar callus) consisting of proembryoids. These are embryolike structures which are bipolar units and germinate into full plantlets under suitable culture conditions.
In dicotyledonous plants totipotent embryogenic cells have been most commonly obtained from explants of embryonic or young seedling tissues. Excised small tissues from young inflorescences (before maturation of floral primordia) are equally effective for the induction of somatic embryogenesis in cultures. Other explants used are the scutellum, young roots, petioles, immature leaf, and immature hypocotyl.
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A high yield of embryogenic calli can also be obtained from isolated fully differentiated mesophyll cells or protoplasts in a defined culture medium. Somatic embryos germinate in situ or when they are excised and cultured individually on a fresh semisolid medium.
The essential requirements for the induction and promotion of somatic embryos are primarily established by the use of suspension cultures. The in vitro development of somatic embryos was first observed in carrot suspension cultures by Steward et al. (1958) although Reinert (1958) was also able to induce somatic embryogenesis in a callus cultured on a semisolid medium.
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The presence of auxin in the medium is generally essential for embryo initiation.
Tissues or calli maintained continuously in an auxin free medium generally do not form embryos; therefore, somatic embryogenesis is achieved in two steps. First, the callus is initiated and multiplied on a medium rich in auxin (2, 4-D, 0.5 mg 1-1) which induces differentiation of localized groups of meristematic cells called embryogenic clumps.
Second, the embryogenic clumps develop into mature embryos when transferred to a medium with a very low level of auxin (0.01-0.1 mg ,-1) or no auxin at all.
Hence a medium with auxin is called a proliferation medium and one without auxin an embryo development medium. Mature embryos develop in embryo development medium. Mostly 2, 4- D has been used to induce somatic embryogenesis.
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A substantial amount of nitrogen, usually in reduced form such as ammonium salts, is required for both embryo initiation and maturation. The source of nitrogen can be in the form of complex addenda such as coconut milk, casein hydrolysate, a mixture of amino acids or single amino acid, and ammonium ions.
MS medium contains high levels of nitrogen in the form of ammonium nitrate. Other items, such as potassium, dissolved oxygen, activated charcoal are also found to playa role in somatic embryogenesis.
In monocotyledonous plants, most of which are agriculturally and medicinally important, the vegetative parts do not readily proliferate in cultures. Hence explants are best taken from embryogenic or meristematic tissues (young inflorescences and leaves).
Germination of the somatic embryo can occur only when it is mature enough to have functional shoot and root apices capable of meristematic growth. High auxin levels can inhibit development and growth of the shoot meristem and often embryos mature when the embryogenic cell suspension is transferred to a medium lacking auxin. The addition of low level of cytokinin (zeatin 0.1 µm) in combination with ABA may prove beneficial for embryogenesis of low density cell cultures.
Various physical factors such as temperature also affect embryo maturation, depending on the requirement of the species. Maturation of somatic embryos proceeds more normally in complete darkness although light seems essential for somatic embryogenesis in some cultures. Somatic embryos germinate on agar medium without growth regulators. Single embryos may profit by the inclusion of low levels of zeatin (0.1 µm) in the medium.
Sometimes loss of morphogenic potential in embryogenic cultures is seen. This may be due to many reasons. Nuclear changes such as polyploidy, aneuploidy and chromosomal mutations in cultured cells may be responsible for the loss of organogenic or embryogenic potential in prolonged cultures. Altered hormonal balance within the cells or tissues may also be associated with decline in the embryogenic potential.
Sometimes the non embryogenic cells of the explant increase under conditions favorable to their growth, resulting in a gradual loss of the embryogenic component during repeated subcultures.
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