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The following points highlight the chemical and other factors affecting somatic embryogenesis.
The chemical factors are: (a) Auxin (b) Cytokinin (c) Gibberellin and (d) Reduced Nitrogen.
(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).
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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 differentiation. After repeated subculture in the same medium, the callus tissue shows a gradual decline in somatic embryogenesis.
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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 observation 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 results. In carrot suspension culture, zeatin (0.1 µM) a type of cytokinin, stimulates embryogenesis 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 embryogenesis.
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 KNO3 produces near-optimal numbers of embryoids. It is, therefore, convenient to use NH+4 in combination 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 supplement 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.
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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.