1 Sep 2020

Somatic Embryogenesis: Factors and Process of Induction

Anjali Singh, MS

As a content and community manager, I leverage my expertise in plant biotechnology, passion for tissue culture, and writing skills to create compelling articles, simplifying intricate scientific concepts, and address your inquiries. As a dedicated science communicator, I strive to spark curiosity and foster a love for science in my audience.

Table of Contents

Somatic embryogenesis is the process of inducing embryo formation in somatic cells of the embryo sac (or cells surrounding it). The idea is based on the concept of totipotency of the plant cells and it demonstrates the two aspects of the plant embryogenesis:

• The process of fertilization can be substituted by an endogenous mechanism.
• Aside from the fertilized egg cells, other cell types of the plant can regain the capability of embryo formation.

The process of somatic embryogenesis doesn’t involve the steps of fertilization (sexual fusion of gametes) and thus, facilitates the rapid large scale propagation of the plants. It also aids in the genetic transformations of plants and acts as a powerful tool for cryo-storage of the embryo and valuable germplasm.

The great potential of somatic embryogenesis and its application for several species, ranging from monocots to dicots (specifically woody and herbaceous species), is now the subject of many studies.

In the article “Somatic Embryogenesis”, a summary of the process is explained with several examples of its applications that have been studied until now.

The present article demonstrates the process of induction of somatic embryos and the factors which affect the process of somatic embryogenesis.

Induction of Somatic Embryogenesis

The induction step involves the reactivation of cells to differentiate and develop embryos. It is achieved by two methods: direct embryogenesis and indirect embryogenesis.

Direct embryogenesis involves the development of embryos directly from the cells of explants, like from the cells of immature embryos. It doesn’t involve any intermediary stages, such as callus formation. The explants of somatic embryogenesis are observed to carry pre-embryogenic determined cells (PEDCs).

Whereas, indirect embryogenesis involves the formation of the somatic embryos by repeating several cycles of cell divisions. It involves an intermediate step of callus growth, so it’s a multistep process.

Cells not carrying PEDCs are induced to differentiate and form the embryo by exposure to several suitable treatments. After this step, cells transform into “induced embryogenic pre-determined cells” (IEDs) that are equivalent to PEDCs.

The induction of somatic embryogenesis has become a routine procedure for some woody species and specifically for some coniferous trees.

Method

Here, a study on the in-vitro induction for somatic embryogenesis, by Widuri, Dewanti, and Sughiharti (2016) is presented.

Material Required:

One-month-old plantlets cut from the 0.5 cm above the base of the stem, MS media, PGR (plant growth regulators) which include 2,4 - Dichlorophenoxyacetic acid ( 2,4-D ), Benzylaminopurine ( BAP ), casein hydrolyzate (as an organic material), the amino acid proline, 3% sucrose, and phytagel 0.25 %.

Procedure:

1. Culture the explants in a sterile container containing media and growth regulators.
2. Place the cultures in dark to induce callus at the temperature ranging from 23℃ - 25℃ for 5 weeks.
3. Separate the embryogenic callus from the induction medium into smaller sections and transfer them to the proliferation medium.
4. Place the cultures in dark at a temperature of 23℃ - 25℃ for 4 weeks.
5. When the callus forms a well-developed shoot-system, transfer it to basic MS media. The cultures should be placed in light at a temperature of 23℃ - 25℃ to induce plant development.

Factors Affecting Somatic Embryogenesis

The process of somatic embryogenesis is a three steps procedure, which includes the induction of embryogenesis, embryo development, and embryo maturation. The last step of the process depends on several factors that are discussed in this section.

1. Explant

The choice of explant depends on the species of plant to be induced for embryogenesis. For most of the plant species, the explants of an immature zygotic embryo work best for somatic embryogenesis (SE).

For example, in conifers, immature zygotic embryo explants are used to induce somatic embryogenesis. In this case, cells in the suspensor region of the zygotic embryo are used for the process.

However, in soybean, cotyledons show better embryogenic response than an immature zygotic embryo. Whereas, in alfalfa, petiole sections from 2-3 young and fully extended leaves are suitable to induce SE.

2. Genotype

The genotypic variation between the plants also affects the process of embryogenesis. And, according to scientists, it may be due to the endogenous level of hormones.

For example, in alfalfa, most of the cultivars have a 10% regeneration capacity. However, the Rangelander variety is found to have a higher growth frequency than others.

Similarly, 500 varieties of rice have been screened for induction of embryogenesis and only 19 were found to have higher responses ranging from 65-100%.

3. Growth Regulators

Auxin: It plays an essential role in the first step of embryogenesis, i.e. the induction step. Almost, all the well studied plants require synthetic auxin for the induction of somatic embryogenesis.

However, some scientists have proved that the induction of embryogenesis in explants is also possible by using a higher concentration of sucrose or varying pH. For example, in carrots, an increase in pH (to 5.6) results in the proliferation of the explants.

All the major cereals or grasses require auxin (2,4-D) in the media, in the concentration ranging from 1-2.5 mg/l. Furthermore, a very high level of auxin results in the inhibition of the embryogenesis in the explants of citrus plants.

Cytokinin: It is an important growth regulator for tissue culture as it induces cell division in the explants. However, several studies have reported that the addition of BAP in the media results in the inhibition of embryogenesis. 

But some studies have also reported that cytokinin promotes the process of embryogenesis. As in the case of coffee where cytokinin is alone enough to induce embryogenesis in the explants.

4. Nitrogen Source

The form of nitrogen used in the media affects the process of embryogenesis in plants. For example, in some plants, reduced nitrogen (such as KNO3) is required to induce the embryogenesis (in carrot), whereas in alfalfa and orchardgrass, ammonia is required for the process.

Several studies have also shown that the presence of amino acids (especially proline and serine/threonine) in the media gives a better embryogenic response in the majority of plants.

5. Polyamines

The process of somatic embryogenesis is also affected by the concentration of polyamines in the explants or the media. Scientists have found that the concentration of polyamines (such as putrescine, spermidine, and spermine), are found to be present in higher concentrations in polyembryonates than the monoembryonates.

However, the relationship between the concentration of the polyamines and the induction of somatic embryogenesis has yet to be established.

References

1. Pais M. S. (2019). Somatic Embryogenesis Induction in Woody Species: The Future After OMICs Data Assessment. Frontiers in plant science, 10, 240. https://doi.org/10.3389/fpls.2019.00240.
2. Widuri I. Laily, Dewanti Parawita, and Sugiharto Bambang (2016). A simple protocol for somatic embryogenesis induction of in vitro sugarcane ( Saccharum officinarum. L) by 2,4-D and BAP. Biovalentia: Biological Research Journal, 2 (1).
3. Jingli Yang, Songquan Wu, Chenghao Li (2013). "High-Efficiency Secondary Somatic Embryogenesis in Hovenia dulcis Thunb. through Solid and Liquid Cultures", The Scientific World Journal. DOI: https://doi.org/10.1155/2013/718754.
4. Zavattieri, M., Frederico, A., Lima, M., Sabino, R., and Arnholdt-Schmitt, B. (2010). Induction of somatic embryogenesis as an example of stress-related plant reactions. Electronic Journal of Biotechnology, 13 (1). DOI: 10.2225/vol13-issue1-fulltext-4.
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