The below mentioned article provides an short notes on Somatic Embryogenesis.
The formation of embryos (embryoids) in plant tissue culture was first reported by Steward (’59) in cell suspension culture of carrot. Formation of embryoids in callus culture of carrot on agar medium was reported by Reinert (’59). Vegetative embryoids have been noticed in tissue culture of various plants, such as, Afiium, Citrus, Cuscuta, Datura, Daucas and Nicotiana.
Embryogenesis has also been observed in several other angiospermic plants, such as, Atropa belladonna, Bromus inermis, Solanum melongena, Ranunculus scleratus etc. and also in some gymnosperms, such as different species of Cycas.
Reinert (’59) observed that when undifferentiated callus was cultured on a medium supplemented with vitamins, amino-acids, the cells became granular. When such cells were transplanted on a medium having low auxin concentration, then many embryoids were formed. These embryoids later produced plantlets.
A suspension of cells from carrot roots when cultured on a medium containing coconut milk then many embryoids are formed. According to Steward coconut milk is required for initiation and development of embryoids. But other scientists do not support this view.
Somatic embryoids may be formed from:
(a) Vegetative cells of a mature plant,
(b) Reproductive cells other than zygote or
(c) Cotyledons, hypocotyl or young plantlets.
According to Sharp (’80) embryoids are formed in two ways:
(a) Directly, without callus formation, from pre-embryonic cells, i.e., the cells that are destined to form the embryo,
(b) After callus formation from induced embryo genic cells, as in carrot.
In carrot root culture it has been observed that single cell should produce a cell aggregate (pro-embryo) first, before embryo initiation. During initiation embryo genic cells have protoplasmic connections with adjacent cells.
Embryo genic cells have certain characteristics. These cells have dense cytoplasm, prominent nucleolus, large nucleus, conspicuous starch grains, high concentration of protein and RNA and have dehydrogenase activity. These cells are distinguished by their staining behaviour.
Embryoid development in tissue culture passes through three stages, namely, globular stage, heart-shaped stage and torpedo stage (Fig. 20). During embryoid formation there is first cyto-differentiation of proembryoid cells followed by occurrence of various developmental stages.
Factors influencing somatic embryogenesis:
(1) Auxin:
In medium having relatively high concentration of auxin embryonal budding or embryonal clumps have been observed. For cell differentiation the medium should contain auxin and reduced nitrogen.
Subsequent development takes place in medium with no auxin or low concentration of auxin and reduced nitrogen. In some plants first and second stages occur in the first medium and plantlet development takes place in the second medium.
(2) Nitrogen:
The ratio of nitrogen to auxin is an important factor controlling embryogenesis. Embryo development can be initiated on White’s medium with low nitrogen content only in absence of auxin. At low nitrogen concentration organic nitrogen is more suitable than inorganic nitrogen.
Substances used as a source of nitrogen are potassium nitrate, ammonium chloride, glutamine, glutamic acid, alanine, urea etc. Endogenous polyamines, such as, putrescine, sperdimine or spermine are required for induction of embryogenesis in wild carrot culture.
(3) Cytokinin:
The role of cytokinin in embryogenesis is not clear. Embyogenesis in carrot cell suspension is stimulated by addition of zeatin in medium lacking auxin but inhibited by the addition of kinetin. Inhibitory effect of exogenous cytokinin may be due to an increase in endogenous cytokinin in growing embryoids.
(4) Activated charcoal:
Presence of activated charcoal in the medium helps embryogenesis in several cases, as in Daucas carota, Hedera helix etc. Activated charcoal may adsorb the inhibitory substances present in the medium.
(5) Age of the culture:
Embryogenesis usually occurs in short-term cultures. With older cultures this ability decreases and ultimately it is completely lost. This may be due to either the inability to synthesise some embryogenetic substances or changes in the ploidy level which may lead to loss of morphogenetic potential.
In carrot culture embryoid formation starts 4-6 weeks after isolation of the tissue. The, optimal embryo genetic potential is reached after 15 weeks, then this potentiality gradually decreases and ultimately lost after 36 weeks. But carrot tissue may regain the embryo genetic potentiality when transferred to a medium containing proper nutrient substances.
Methods of embryogenesis from cell suspension culture of carrot root:
Carrot roots are cultured and callus is obtained. From an actively growing callus explants are taken for suspension culture on Murashige and Skoog’s medium supplemented with sucrose, 0.2 mg/1 zeatin and 1 % agar.
On this auxin free medium embryoids are formed. After the embryoids have reached the torpedo stage, they are transferred to filter paper bridges on tubes containing Murashige and Skoog’s medium with 2% sucrose and 0.2 mg/1 kinetin. Plantlets formed are later transferred to soil under humid condition.
Significance:
By somatic embryogenesis many plants are produced very rapidly. By this method a desirable plant can be multiplied very quickly.