Somatic Embryogenesis: Process and Applications | Plants

In this article we will discuss about: 1. Process of Somatic Embryogenesis 2. Embryo Maturation and Synchronisation 3. Cultural Conditions 4. Recurrent Embryogenesis and Mass Production 5. Applications.

After fertilization, zygote is transformed into adult status through a series of embryogenic processes. Despite the same genetic constituents, somatic cells on the other hand, do not reorient towards embryo production. However, isolated somatic cells under in vitro conditions have the potential to develop into embryo under the influence of growth factors.

Somatic cells are able to develop into whole plant through the stages of embryogenesis without gametic fusion. Therefore, somatic embyros are non-zygotic embryos originated from sporophytic cells. Somatic embryo production is either direct or indirect in vitro. Somatic embryos may be direct when embryonic cells develop directly from the explants’ cells or indirect when developed through the callus.

Plant cells undergoing somatic embryogenesis are either pro-embryonic determined cells (PEDC) or induced embryogenic determined cells (IEDC). There have been reports on the induction of somatic embryos frequently from various tissues like seedlings, shoot meristem, young inflorescence and zygotic embryos. In addition, other tissues such as root, nucellus has also yielded somatic embryos.

The favorable responses of a few of the above tissues actually contain proembryogenic determinant cells (PEDC) or these cells may require minor reprogramming to enter embryogenic state. The first report on the production of somatic embryos in carrot suspension cells was published by Steward and co-workers in 1958. Thereafter reports were flooded on the production of somatic embryos in plants.

Process of Somatic Embryogenesis:

Significance of Auxin:

Reprogramming of somatic cells and its entry into the embryogenic status requires ex­tensive proliferation through unorganized callus cycle and exposure to high doses of synthetic auxin such as 2, 4-D or picloram. Somatic embryo induction can also be accomplished by plasmolysis of explant cells.

The significance of auxin for embryo induction status from vegetative cells and tissue was recognized as the prime controlling factor. This is based on a critical assessment in species like Daucus carota, Atropa belladona and Ranunculus sceleratus. Transformation of embyrogenic cells into the callus system due to the differentiation of single cell is followed by the appearance of dense cytoplasm, prominent nucleus and high profiles of organelles.

These groups of small densely packed cytoplasmic cells arise by internal division. These groups of cells constitute pro-embyros which can develop into globular embryos. The formation of a mature embyro and plantlet via heart and torpedo shaped stages may proceed undisturbed even when exogenous auxin remains present at lowest concentration in the later development.

The later process in the prevalent media condition however, may disturb further establishment of embryogenesis unless auxin is completely omitted. It was even evidenced that embryogenic process may be completely arrested during transition of embryo to plantlet. Therefore auxin is reduced or entirely withdrawn once such anomaly appears during culture.

In date palm tissue culture, liquid media enriched with low amount of plant growth regulator resulted in the differentiation of large number of somatic embryos. High concentration of auxin may not encourage embryo formation. Therefore, two distinct conclusions can be drawn by the role of auxin in entire embryogenic episode.

First, induction of cells with reprogrammed embryogenic competence under the influence of auxin. Second one is directing embryogenic cells to undergo complete development by withdrawing auxin from the media. Low level of endogenous auxin can equally determine embryo induction.

Auxin deprivation acts as a development switch from nonpolar embryogenic units to induce somatic embryogenesis in maize. This developmental switch is accompanied by cytoskeletal rearrangements in embryogenic cells. Whole somatic embryogenetic process may derail the establishment of polarity if exogenous auxin is supplied.

One of the negative factors implicated in somatic embryogenesis is the pro­duction of ethylene in presence of auxin for a considerable period of time in the culture media. Production of ethylene in turn elevates the activity of enzymes, probably, cellulase and pectinase which degrade pectin compounds and consequently disturb establishment of polarity by reduc­ing cell to cell interaction and contact due to separation.

Role of 2, 4-D in particular, for the induction of somatic embryogenesis is exemplary. Literature survey has shown that this synthetic auxin is very often suitable in inducing somatic embryogenesis in most of the species. Another synthetic auxin NAA has been found to be suit­able for somatic embryo induction.

However, the role of phytohormones in somatic embryo induction is highly a complex process and varies depending on plant species as well as its en­dogenous concentration. Under no circumstances, gibberellic acid is useful for somatic embryo induction. But its role has been implicated in the maturation of somatic embryos.

Role of Reduced Nitrogen:

The embryogenic competent cells seem to have preference for high salt strength and specific nitrogen source. This was considered to be a second pre-requisite for somatic embryo induction after auxin. The reduced form of nitrogen, ammonia, provides triggering factors for embryogenesis.

Similarly, nitrogen in the form of casein hydrolysate can equally contribute in the stimulation of somatic embryos and has been critically assessed in carrot as a model plant. Presence of proline and serine, capable of stimulating somatic embryo induction was reported in carrot plant.

Addition of reduced nitrogen, ammonium ion (NH₄⁺ salt) or amino acids into the media is conductive for embryogenesis after shifting callus from auxin to auxin free media. It is however, concentration of auxin and nitrogen rather than critical concentration of reduced ni­trogen which is crucial in empowering embryogenesis.

High frequency of somatic embryogenesis was achieved in cucumber plant. Addition of diazuron and sucrose treatment (3-6%) exerted positive effect on the relative position of somatic embryo induction. Addition of copper sulphate in the media induces high frequency somatic embryo induction.

Similarly, thiadiazuron when supplemented in the medium induced shoot organogenesis at low concentration and somatic embryogenesis at high concentration. Enhanced somatic embryo production and maturation into normal plants in cotton was achieved when calli cultured on half strength MS media.

A thorough examination of the role of reduced nitrogen ammonia shows that embryo formation is promoted when as little as 0.1 mM ammonium chloride is supplied to nitrate me­dia. Embryogenesis is promoted by 40 mM potassium nitrate and 30 mM ammonium chloride as optimum concentration.

Glutamine and alanine can serve as sole nitrogen source for the growth and embryo formation. Although nitrate is required for embryogenesis on several in­stances, ammonium alone can produce embryo in carrot suspension culture, provided pH of the medium containing 10mM ammonium chloride and 20 mM potassium chloride was controlled at pH 5.4.

Level of dissolved oxygen has some role to play in somatic embryogenesis at least in carrot plant where embryogenesis takes place only below critical level of dissolved oxygen (i.e., above 1.5 ppm). Higher level favors rhizogenesis. Addition of activated charcoal into the culture media can promote embryo induction by adsorbing inhibiting substances produced by tissue.

Embryo Maturation and Synchronisation:

Studies on embryo germination process shows that embryo development completes without any anomalies in the absence of auxin in the media. However, any abnormalities due to endogenous hormones can be avoided by supplementing balanced concentrations of abscisic acid (ABA), zeatin, and GA₃. Addition of charcoal may increase the maturation of somatic embryo.

Presence of charcoal in the media reduces the level of auxin like IAA due to its binding effect. Somatic embryo maturation can be enhanced by subjecting to osmotic desiccation. Sucrose is generally used at different concentrations to achieve embryonic growth and maturation. This is achieved by providing sucrose concentration between 4 and 6%.

In certain species, progressive increase in sucrose concentration upto 4% is required for maturation, which consequently produces vigorous plantlets. Similarly, imposition of temporary desiccation before embryo germination facilitates conversion to plantlets. Imposition of desiccation can be progressed by placing somatic embryos in empty petridish and incubated at desiccated condition for 2-3 weeks and some plants upto several weeks.

Somatic embryos, when shriveled to 50% of their original volume rapidly imbibe water when rehydrated by transfer to media. The whole exercise of desiccation in embryo is to influence metabolic process for germination. Somatic embryos when subjected to show desiccation, it stimulates the production of high frequency of shoot regeneration.

Imposition of desiccation improves conversion to plantlets several times the frequency of non-desiccated embryos. In Alfalfa culture, somatic embryos have been trained to withstand desiccation by treating them with ABA at the torpedo stage. ABA treatment can promote the development of cotyledons and block the production of embryo clusters.

Cultural Conditions of Somatic Embryogenesis:

High light intensity can influence the process of somatic embryogenesis. However, cul­tures were incubated under both light and dark periods. Early maturation takes place more predominantly under complete dark conditions.

Reports on the influence of temperature on somatic embryogenesis are scarce. In citrus nucellus culture, embryogenic potential drops when the temperature was reduced from 27°C to 12°C. Similarly, conditioning of somatic embryos by cold treatment can escape dormancy and facilitate development.

Recurrent Embryogenesis and Mass Production of Somatic Embryogenesis:

The primary somatic embryo when fails to undergo maturation may enter continuous successive cycles of embryos. Certain specific superficial cells of the hypocotyl or cotyledon exhibit this tendency in provoking successive cycles of embryos or in other words continuous production of supernumerary embryos from somatic embryos itself.

This phenomenon is also known as secondary embryogenesis, recurrent embryogenesis, repetitive or accessory embryogenesis (Fig. 8.1). Recurrent embryogenic cycle can be maintained in culture by the removal of growth regulators and cycles can be spontaneous as this was evidenced in Alfalfa (Medicago sativa).

Recurrent embryogenesis cycle can be made spontaneous by locking the development of so­matic embryos particularly at proembryogenic status, beyond which they cannot proceed to develop. This can be accomplished by initial exposure to very high concentration of 2, 4-D upto 40 mg/L for brief period followed by exposure to a lowest concentration (3-5 mg/L).

This high concentration of auxin treatment may be involved in reprogramming of cells and reinduce embryogenic competence. Repetitive embryogenesis may be a serious problem during spontane­ous cycles of somatic embryo production when germination and further development is required.

Gene Expression Programme in Somatic Embryogenesis:

One of the most striking features of somatic embryogenesis is the successful crackdown on RNA expression in embryogenic and nonembryogenic tissues. Several striking similarities were cited in the gene products expressed in embryogenic and nonembryogenic cultures. The tissue culture conditions are typically defined as nonembryogenic. The pattern of gene expres­sion between embryogenic and nonembryogenic systems exhibit least diversity about RNA ex­pression profiles.

Limited number of changes has been recorded in protein expression pattern during somatic embryogenesis. Similarly, changes in mRNA populations take place during tran­sition from nonembryogenic to various embryo stages. Removal of auxin from the media during embryo induction triggers new profiles of gene expression that are eventually coupled to ob­serve morphogenetic events.

Applications of Somatic Embryogenesis: 

Micro-Propagation Industries:

One of the most promising applications of somatic embryogenesis is large scale propagation of somatic embryos, which shows several advantages such as innumerable number of embryo production (60,000-70,000 embryos per litre of media), presence of both root and shoots meristems, easy to scale up and convert them into seedlings efficiently as far as commercial significance is considered. Somatic embryos are genetically well programmed to make a complete plant. Thus, unlike other micro-propagation systems, somatic embryogenesis avoids certain stages of micro-propagation particularly, the rooting stage.

Synthetic Seed Production:

Synthetic seeds or artificial seeds are the somatic embryos encapsulated by gel entrap­ment solution. Artificial seeds are generally produced in plant species which exhibit seed steril­ity and difficulty or slow phase of vegetative propagation.

This can be prepared by placing somatic embryos in alginate slurry (2%) as gel entrapment matrix and subsequently trans­ferred to calcium chloride (100 mM) solution to form beads in which embryos get entrapped. Artificial seeds can be stored at 4°C for a considerable period of time and used as efficient system for germplasm conservation. For regeneration, seeds can be placed in culture media or in sterile soil to facilitate germination and seedling development (Fig. 8.2).

Embryo Cloning:

Repetitive embryogenesis often provides innumerable number of somatic embryos, which in turn is useful in the mass production of plant propagules. Several embryo specific metabolites like seed storage proteins and lipids of industrial value can be recovered. Lack of seed tissue surrounding somatic embryos proves significant advantage for certain lipids such as α-linolenic acid present at high level in Borage seeds.

This lipid is of high commercial significance in the treatment of atopic eczema. Surprisingly, somatic embryos as an analogue of zygotic embryo also synthesize the same amount of α-linolenic acid. Similarly, jojoba plant contains high qual­ity industrial lubricant in their seeds.

Somatic embryos obtained from zygotic embryos as the explant possesses waxes identical to that of zygotic embryos. In addition, novel metabolites can be produced in somatic embryos throughout the season.

Somatic Embryos in Gene Transfer:

Somatic embryos are an ideal system for gene transfer process. This particular approach can avoid protoplast mediated regeneration of transformed plants which generally requires additional care. Moreover, protoplast mediated regenerated plants can exhibit genetic variation. Since somatic embryos maintain genetic stability, regenerated plants are not susceptible for somaclonal variation.

Somatic embryos can be transformed by incubating them in Agrobacterium solution or subjected for particle bombardment. Embryo cloning by recurrent approach is well suited for direct gene transfer to the mass of somatic embryos. The stably transformed somatic embryos can be farther subjected for recurrent embryogenic cycle to procure millions of transgenic plants.