Let us make an in-depth study of the meaning, history, principles, protocols and importance of somatic embryogenesis.
About Somatic Embryogenesis:
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.
What is Embryo genic Potential?
The capability of the somatic plant cell of a culture to produce embryoids is known as embryo genic potential.
What is Embryoid?
Embryoid is a small, well-organised structure comparable to the sexual embryo, which is produced in tissue culture of dividing embryo genic potential somatic cells.
Brief Historical Background:
J. Reinert (1958-59):
Reported his first observations of in vitro somatic embryogenesis in Daucus carota.
F. C. Steward, M. O. Mapes and K. Mears (1958):
Also reported the somatic embryogenesis in carrot from freely suspended cells and emphasized the importance of coconut milk for in vitro somatic embryogenesis.
N. S. Rangaswamy (1961):
Studied in detail the somatic embryogenesis in Citrus sp.
R. N. Konar and K. Nataraja (1969):
Studied the somatic embryogenesis of Ranunculus sceleratus using various floral parts (including anthers) as well as somatic tissues in culture.
P. V. Ammirato (1974):
Reported the effect of abscisic acid on the development of somatic embryos from cells of Carum carvi.
H. Lang and H. W. Kohlenbach (1978):
Demonstrated the ability of mechanically isolated, fully differentiated mesophyll cells of Macleaya cordata to yield an embryogenic callus.
B. V. Conger, G. E. Hanning, D. J. Gray and J. K. McDaniel (1983):
Obtained direct embryogenesis from leaf mesophyll cells of orchard grass (Dactyhs glomerata L.) without an intervening callus tissue.
Principles of Somatic Embryogenesis:
Somatic embryogenesis may be initiated in two different ways:
1. In some cultures somatic embryogenesis occurs directly in absence of any callus production from “pro-embryo genic determined cells” that are already programmed for embryo differentiation (Fig 8.1). For instance, somatic embryos has been developed directly from leaf mesophyll cells of orchard grass (Dactyhs glomerata L.) without an intervening callus tissue.
Explants, made from the basal portions of two innermost leaves of orchard grass were cultured on a Schenk and Hildebrandt medium supplemented with 30 µM 3, 6-dichloro-O-anisic acid (dicamba). Plant formation occurred after sub culturing the embryos on the same medium without dicamba (Conger et al., 1983).
2. The second type of somatic embryo development needs some prior callus formation and embryoids originate from “induced embryo genic cells” within the callus tissue.
In most of the cases, indirect embryogenesis occurs. For indirect somatic embryogenesis where it has been induced under in vitro condition, two distinctly different types of media may be required—One medium for the initiation, of the embryonic cells and another for the subsequent development of these cells into embryoids.
The first or induction medium must contain auxin in case of carrot tissue and somatic embryogenesis can be initiated in the second medium by removing the hormone or lowering its concentration. With some plants, however, both embryo initiation and subsequent maturation and subsequent maturation occur on the first medium and a second medium is required for plantlet development.
In some cases, a given culture may differentiate the embryo genic cells, but their further growth may be blocked by an imbalance of nutrition in the culture medium. According to Kohlenback, (1978), abnormalities known as embryonal budding and embryo genic clump formation may occur, if relatively high level of auxin is present after the embryo genic cells have been differentiated.
Embryoids are generally initiated in callus tissue from the superficial clumps of cells (primordia) associated with enlarged vacuolated cells that do not take part in embryogenesis. The embryo genic cells are generally characterised by dense cytoplasmic contents, large starch grains, a relatively large nucleus with a darkly stained nucleolus. In suspension culture, embryoids do not form suspended single cell, but form cells lying at or near the surface of the small cell aggregates (Fig 8.2).
Each developing embryoid of carrot passes through three sequential stages of embryo formation such as globular stage, heart-shape stage and torpedo stage (Fig 8.3). The torpedo stage is a bipolar structure which ultimately gives rise to complete plantlet. The culture of other plants may not follow such sequential stages of embryo development.
In general, somatic embryogenesis occurs in short-term culture and this ability decreases with increasing duration of culture. But there are some exceptional cultures where embryogenesis has been reported from the callus tissue maintained over a period of year. According to Smith and Street, (1974), changes in ploidy of the cultured cell may lead to loss of embryo genic potential in long term culture. The loss of embryo genic potential in long term culture may also result from loss of certain biochemical properties of the cell.
In callus culture or in suspension culture, embryoid formation occurs asynchronously. Some progress has been made in inducing synchronization of somatic embryogenesis in cell suspension culture. A high degree of synchronization has been achieved in a carrot suspension culture by sieving the initial cell population.
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.
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.