Somatic Embryogenesis: Types, Protocol and Importance | Plant Biotechnology

In this article, we will discuss about:- 1. What is Somatic Embryogenesis? 2.Types of Somatic Embryogenesis 3. Requirements 4. Protocol and 5. Importance of Somatic Embryogenesis

1. What is Somatic Embryogenesis?

In plant tissue culture, the developmental pathway of numerous well-organised, small embryoids resembling the zygotic embryos from the embryo genic potential somatic plant cell of the callus tissue or cells of suspension culture is known as somatic embryogenesis.

2. Types of Somatic Embryogenesis:

1. Direct somatic embryogenesis: It is the formation of embryo directly from a single cell without an intervening callus stage. Although embryo arises by means of the dedifferentiation of a differentiated cell within the explant.

2. Indirect somatic embryogenesis: It is the process of embryogenesis via intervening callus. The embryogenic callus is composed of young embryos (pre-embryogenic masses or globular embryos) and their further development depends upon the duration of the application of the inductive stimulus. If the period is relatively shorter the process will be direct and if it is long then the process will be indirect

3. Requirements for Initiation of Somatic Embryogenesis

i) Explants

Any part of plant which is directly used to initiate process of tissue culture is known as explant and the plant from which the part has been taken is called mother plant. In Dicot different plant parts such as; young seedling tissues or embryogenic tissue, inflorescence, young roots, petiole, immature leaf, immature hypocotyle of about 3 weeks can be used for initiation of somatic embryogenesis. In Monocots following explants are used

• Zygotic embryo - young seeds 10-15 days after pollination
• Young inflorescence - 1-2cm in length on sterilized medium
• Young leaf - of unexpanded young seedling 1-2 mm

ii) Other basic requirements

In other basic needs the followings are of most importance;

Auxin supply: Auxins are type of hormones and are essential for proper growth of plant or plant parts. In somatic embryos development process auxin play role in two steps. First the callus is initiated and multiplied in medium with high auxin (2,4-D) supply. Secondly this localized group of meristimatic cells called embryogenic clumps (ECs) is differentiated on proliferation media to develop into mature embryos in low auxin (0.01-0.1mg/L) or no auxin supply at all. Among auxins 2,4-D is commonly used but NAA and IBA use vary from plant to plant. In some cases zeatin has also been reported to use in development of somatic embryos.

Nitrogen Source: Nitrogen supply is very necessary in somatic embryogenesis as it is required for initiation and maturation of somatic embryos. It is used as in reduced form such as ammonium salt. For example in carrot high conc. of nitrogen (in the form of ammonium nitrate) is used in MS medium for development of somatic embryos. Sometimes NH4Cl and NH4NO3 both are used for somatic embryogenesis in number of cultures. 

4. Protocols for Inducing Somatic Embryogenesis in Culture:

The plant material Daucus carota repre­sents the classical example of somatic embryo- genesis in culture.

The protocol is described below:

1. Leaf petiole (0.5-1 cm) or root segments from seven-day old seedlings (1 cm) or cam­bium tissue (0.5 cm3) from storage root can be used as explant. Leaf petiole and root segment can be obtained from aseptically grown seedlings (Cambium tissue can be obtained from surface sterilized stor­age tap root 2. Following aseptic technique, explants are placed individually on a semi-solid Murashige and Skoog’s medium containing 0.1 mg/L 2, 4-D and 2% sucrose. Cultures are incubated in the dark. In this medium the explant will produce sufficient callus tissue.

3. After 4 weeks of callus growth, cell suspen­sion culture is to be initiated by transferring 0.2 gm. of callus tissue to a 250 ml of Erlenmeyer flask containing 20-25 ml of liquid medium of the same composition as used for callus growth (without agar). Flasks are placed on a horizontal gyratory shaker with 125-160 rpm at 25°C. The presence or ab­sence of light is not critical at this stage.

4. Cell suspensions are sub-cultured every 4 weeks by transferring 5 ml to 65 ml of fresh liquid medium.

5. To induce a more uniform embryo popula­tion, cell suspension is passed through a se­ries of stainless steel mesh sieves. For car­rot, the 74 µ sieve produces a fairly dense suspension of single cell and small multiple clumps. To induce somatic embryogenesis, portions of sieved cell suspension are trans­ferred to 2, 4-D free liquid medium or cell suspension can be planted in semi-solid MS medium devoid of 2, 4-D. For normal em­bryo development and to inhibit precocious germination especially root elongation, 0.1- 1 µM ABA can be added to the culture medium. Cultures are incubated in dark.

6. After 3-4 weeks, the culture would contain numerous embryos in different stages of de­velopment.

7. Somatic embryos can be placed on agar me­dium devoid of 2, 4-D for plantlet develop­ment.

8. Plantlets are finally transferred to Jiffy pots or vermiculite for subsequent development.

5. Importance of Somatic Embryogenesis:

The potential applications and importance of in vitro somatic embryogenesis and organo­genesis are more or less similar. The mass pro­duction of adventitious embryos in cell culture is still regarded by many as the ideal propagation system. The adventitious embryo is a bipolar structure that develops directly into a complete plantlet and there is no need for a separate root­ing phase as with shoot culture.

Somatic em­bryo has no food reserves, but suitable nutrients could be packaged by coating or encapsulation to form some kind of artificial seeds. Such ar­tificial seeds produce the plantlets directly into the field. Unlike organogenesis, somatic embryos may arise from single cells and so it is of special significance in mutagenic studies.

Plants derived from asexual embryos may in some cases be free of viral and other pathogens. For an example, Citrus plant propagation from embryo genic callus of nuclear origin are free of Virus. So it is an alternative approach for the production of disease-free plants.