Factors Affecting Somatic Embryogenesis: Chemical and Other Factors

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).

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.

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.

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.