Somatic embryogenesis

 

What is somatic embryogenesis? Discuss the protocol to induce somatic embryogenesis. What are the factors affecting this phenomenon? 

Somatic embryogenesis:-

      Normally embryo is formed after fertilization from the single cell zygote. In plant tissue culture, the developmental pathway of numerous, well organized, small embryogenic potential somatic plant cell of the callus tissue or cells of suspension culture is known as somatic embryogenesis.

Protocol for somatic embryogenesis:-

       Somatic embryogenesis may be of two types- direct and indirect. In case of direct embryogenesis, cells of explants undergo direct development from proembryonic determines cells, in absence of callus proliferation. On the contrary indirect embryogenesis, cells of explants first undergo callus polyferation and then embryos develop within the callus tissue from somatic embryogenic cell.

       For indirect somatic embryogenesis two distinctly types of medium may be required, one medium for the initiation of the embryonic cells and another for the subsequent development of these cells in the embryoids. The protocol for somatic embryogenesis in culture is given below. In this method the plant material Daucus carota is taken as classical example

(i) The leaf petiole (0.5-1cm) or root segments from 7day cold seedlings (1cm) or cambium tissue from storage root can be use as explants. Cambium tissue can be obtained from surface sterilize storage tap root.

(ii) The explant is surface sterilized through ethanol for 30sec.and then by sodium hypochloride and finally wash several times by distilled water. Then explants are placed individually on a semisolid MS medium containing 0.1mg/lt. 2,4-D and sucrose (2%). These cultures are incubated in the dark. In this medium the explants produce sufficient callus tissue.

(iii) After 4 weeks of callus growth, cell suspension culture is to be initiated by transferring a little of callus tissue to a sterilized flusk containing 20-25 ml of liquid medium of the same composition as used for callus growth. Such flusks are placed on a horizontal shaker with 125-160 rpm at 25°c. the presence or absence of light is not critical at this stage.

(iv) Cell suspensions are subcultured every 4 weeks by transferring 5 ml. to 65 ml. in fresh liquid medium.

(v) To induce a more uniform embryo population, cell suspension is passed through a series of stainless still sieve with pore size 74 µ to induce somatic embryogenesis. Portion of sieve cell suspension are transferred to 2,4-D free liquid medium or cell suspension can be plated in semisolid MS medium devoid of 2,4-D.

(vi) After 3-4 weeks the culture would contain many embryos in different stages of development. Somatic embryos are now saptated and are transferred to new medium of same composition for plant let development.

(vii) Plant lets are finally transferred to vermiculite for subsequent development.

Factors affecting somatic embryogenesis:-

       The developmental process of somatic embryogenesis is influenced by some chemical and physical factor. These are described below-

(i) Auxin:-

       Auxins, especially the synthetic auxin 2,4-D, appear to be required for embryogenesis. In Daucus carota auxins are required for embryo induction, i.e. proliferation of the callus tissue and for the induction of embryogenic potential cells. But it has adversely affect on embryo development. In Citrus sinensis, the callus tissue is initial from necellar 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.

       Thus it is suggested that a minimal level of auxin is essential  for the induction of embryogenic potential cells within the callus culture but for the organization and maturation of embryos from embryonic potential cell, auxin does not play any positive role.

(ii) Cytokinin:-

       The effect of cytokinin in somatic embryogenesis is not experimentally established because of conflicting results. In carrot suspension culture zeatin- a type of cytokinin stimulates embryogenesis. But the process is inhibited by kinetin or benzylaminopurine- another type of cytokinin. The development of embryonic cell depends on the auxin:cytokinin ratio.

(iii) Gibberellin, Ethylene and ABA:-

       Ethylene suppresses embryogenesis, abscisic acid suppresses abnormal development of embryos, where as gibberellins produce an increase in frequency of abnormal embryo development. Abscisic acid also imparts dormancy and helps in the formation of cotyledonary stage somatic embryo.

(iv) Reduced nitrogen:-

       Reduced nitrogen is required for embryogenesis. In carrot culture, the addition of ammonium chloride to embryogenic medium, containing KNO₃, produces many embryois. Thus it is suggested that ammonium and nitrate ions are effective for somatic embryogenesis. It also controls the pH balance (medium brought to pH 5.5).

(v) Dissolved oxygen:-

      In liquid cultures, dissolved oxygen can play a critical role in differentiation. Carrot cells produce somatic embryos below 16% dissolved oxygen while above 16% oxygen levels roots are produce.

(vi) Charcoal:-

       The medium supplemented with activated charchol has facilitated embryogenesis in several culture. The induction of embryogenesis is achieved successfully by the addition of charchol when auxin depletion in the medium fails to produce the desired results.

(vii) Other factors:-

       Except the above factors, some amino acids, sucrose and some volatile oils etc affect the embryogenesis. Beneficial effect of certain amino acids like proline and glutamine has been established. Increasing the osmotic concentration by increased sucrose levels or by addition of mannitol or sorbitol, has shown to affect the embryo development. In Citrus some volatile and some non volatile substance inhibit embryogenesis.