Factors Affecting the Embryogenesis
1. Chemical Factors:
i) Auxins:
Somatic embryogenesis in carrot is a classical example. It is two step process. The carrot cells first develop into a callus tissue in the medium containing the auxin namely2, 4-D (0.5 to 1mg/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 namely 2, 4-D (0.5 to 1mg/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 containing 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 embryogenic potential cells. In second step, auxin is no longer required for the embryogenic 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 , NAA and IBA have also been used in other culture system for induction of embryogenic potential cells.
In Citrus sinensis, callus tissue is imitated from the nuclear tissue in the medium containing IAA and kinetin. Such callus when transferred to auxin-free medium causes the induction of embryogenesis. Non – requirement of auxin in medium during second step may be probably due to synthesis of adequate amount of both auxin and cytokinins which they required for growth and somatic embryogenesis.
A minimum of cytokinins in embryogenesis is somewhat obscure because of conflicting results. In carrot suspension culture, zeatin a type of cytokinins, stimulates embryogenesis when the cells are subcultured in auxin –free –medium. But the process is inhibited by the addition of either kinetin or BAP to the medium. The inhibitory effect of cytokinins may be due to selective stimulation of cell of the culture. Stewart et.al ( 1964) also reported the importance of coconut milk for somatic embryogenesis.
iii) Gibberellins:
Gibberellin has no positive effect. In carrot and citrus, gibberellin inhibit somatic embryogenesis.
iv) Reduced Nitrogen:
Substantial amount of reduced nitrogen (NH+4) are required for embryogenesis. In carrot culture, addition of NH4 to embryogenic medium already containing KNO3 produces near-optimal numbers of embryoids. It is therefore convenient to use (NH4) in combination with NO3- . But no other forms of inorganic reduced nitrogen have been as effective as NH4 for somatic embryogenesis.
Glutamine, glutamic acid, urea and alanine are found to partial replace NH4CL as 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 cultures. The induction of embryogenesis is achieved successfully by the addition of charcoal when auxin depletion in the medium fails to produce the desired result. It has been suggested that charcoal may absorb a wide variety of inhibitory substances as well as hormone.
Optimum 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.