Signaling Overview of Plant Somatic Embryogenesis

1Higher plant embryogenesis is divided conceptually into two distinct phases: early morphogenetic processes that give rise to embryonic cell types, tissues, and organ systems, and late maturation events that allow the fully developed embryo to enter a desiccated and metabolically quiescent state. Embryogenesis is the process by which embryo formation is initiated, either from a zygote (zygotic embryogenesis, ZE) or from somatic cells (somatic embryogenesis, SE). ZE is carried out after the fusion of gametes. However, the formation of asexual embryos can be induced in vitro from cells that come from an explant of vegetal tissue. The SE process also occurs in nature. Under certain environmental conditions such as heat and drought, the plant Kalanchoë produces, around their leaves, small bipolar structures, which develop later in plantlets. There are several other paths leading to the formation of an embryo. For instance, apomictic embryogenesis takes place in the seed primordium (ovule) and the embryos produced are genetically identical to the mother plant. Microspores can also produce embryos, and the cells of the suspensor can change their identity to embryogenic cells when the original embryo loses its capacity to develop.

2Somatic embryogenesis represents a complete model of totipotency and involves the action of a complex signaling network, as well as the reprogramming of gene expression patterns that are regulated in a specific way. This gene regulation usually is in response to exogenous stimuli produced by the use of plant growth regulators (PGR) or certain stress conditions, mainly low or high temperature, heavy metals, osmotic shock or drought. The induction of SE in vitro can be accomplished through two pathways. When SE is direct, somatic embryos are formed at the edge of an explant; when it is indirect, SE occurs through the proliferation of a disorganized and dedifferentiated tissue called callus.

3Somatic embryogenesis has several biological and scientific advantages. For instance, it has the potential for the improvement of plants of commercial importance, as well as for the study of the genetic and physiological changes that are related to the fate of a plant cell. Until now, most studies have examined the mechanisms involved in the induction of the SE process using model plant species, such as carrot, alfalfa, corn, and rice. However, other species, such as Arabidopsis thaliana and Gossypium hirsutum, have been used to study the signaling pathways of the PGR action leading to the development of plant cells.

4Once the somatic cells are induced to generate cells with embryogenic capacity, the new cells can form structures capable of regenerating a complete plant. System suspensors are very noticeable in gymnosperm somatic embryos. However, in many angiosperms, suspensors are either absent or strongly reduced due to the absence of the hypophyseal cell.

5It is unclear how cells initiate embryo formation. Nonetheless, it has been established that an irregular distribution of auxins must be established to initiate embryo formation. This asymmetrical auxin distribution results from differential transport (; Figure 1). In the case of ZE, an asymmetric cell division occurs, whereas in SE this is often not observed. An asymmetric mitotic division of the zygote produces two different cells: one cell gives rise to the suspensor and the other to the embryo proper. At the octant and globular stage, protoderm formation and primordial initiation takes place. The differential transport and asymmetrical auxin distribution continue during these stages, giving rise to the different tissues that will form the embryo. The transportation and accumulation of auxin produce the interaction with other factors, such as cytokinins (CKs), which leads to the expression of specific genes.

6Although there is a morphological resemblance between somatic and zygotic embryos, their development is distinctive based on plant classification (angiosperms and gymnosperms). It is considered that zygotic embryos are nourished via the phloem tissue, whereas somatic embryos use an exogenous supply of carbohydrates and their morphological stages occur without vascular tissue connection.

7Theoretically, plant development can be divided into two different phases: (1) embryogenesis sensu stricto, which begins with the formation of the zygote and concludes at the cotyledonary stage, and (2) the maturation of the seed. The somatic and zygotic embryo developmental stages are divided into two main metabolic phases. The first is at a morphogenetic level, where the meristem activity is triggered at a physiological level and the process of growth, storage and maturation is initiated. The second is a metabolic stage that is characterized by biochemical activities and the preparation for desiccation to complete the seed formation process. In this last phase, somatic embryos achieve both morphological and physiological maturity, which guarantees satisfactory post-embryonic performance. Therefore, the conversion potential is considered to be programmed during embryo maturation. However, somatic embryos do not require desiccation.

8Somatic embryo development involves similar stages to ZE, such as the globular-shaped, heart-shaped, torpedo-shaped, and cotyledonal stages in the case of dicotyledonous species, and globular, scutellar, and coleoptile stages in the case of monocotyledonous species. Once the somatic embryos reach the cotyledonary stage, they initiate a shoot meristem, and seedling growth begins.

9Understanding the physiological and molecular mechanisms by which the induction (direct or indirect) of SE occurs is a crucial step for its manipulation. Several factors can induce SE. The conditions of the culture medium, the high concentrations of PGRs, and the wounding of explant are other types of stress that can cause plant cells to change their cellular and molecular programs. The type of explant, the age and the genotype of the mother plant, the physiological conditions of the incubation, and the cellular density in the case of suspension cultures, as well as the generation of homogeneous cell aggregates, are factors that must be considered in order to produce the acquisition of embryogenic potential.

10The source of nitrogen, as well as its concentration in the culture medium, has been shown to be an essential element for the induction of SE. In different plant species, such as Cucurbita pepo, Medicago sativa, Coffea arabica, and Daucus carota, it has been determined that both nitrate and ammonium content in the culture medium have a significant effect on the response of the explants to the induction of SE. It has been proposed that stress is the switch that stimulates cellular reprogramming toward an embryogenic path. However, the mechanism by which the nitrogen sources participate in the induction of embryogenic potential remains unknown.

11In plant culture systems, the addition of PGR to the culture medium plays an important role in inducing cell differentiation, in particular during the induction of SE. Most of the SE process depends on the concentration and kind of PGR used for each culture. Different plant species, such as C. canephora, A. thaliana, and Musa spp. responded successfully to the SE induction using different explants, conditions, and concentrations of PGR.

12Many species that are able to produce somatic embryos from cell suspension cultures require the addition of auxins in the culture medium. The use of 2, 4-dichloroacetic acid (2, 4-D) has an essential role in the induction of SE and the initial stages of development of the somatic embryos. For example, the productivity for embryogenic date palm crops increased 20 times by adding a low concentration of 2, 4-D. The use of auxins modified their endogenous metabolism in a significant way; for example, in carrots, the use of 2, 4-D in the culture medium induces an embryogenic response that is associated with the increase of the endogenous levels of indole-3-acetic acid (IAA). The pre-treatment of plants before the induction of SE in C. canephora also modified the endogenous metabolism of IAA.

13Other PGRs, such as CKs, also participate in the development of the plants, promoting the formation of buds, delaying the aging of the leaves and, together with the auxins, stimulating cell division; both regulators are known to act synergistically. A high ratio between CKs and auxins stimulates the formation of shoots while that a low ratio induces the regeneration of roots and the proper establishment of meristems in Pisum sativum. These two PGR can act either synergistically or antagonistically during the induction of SE. Recent studies using synthetic reporter genes such as DR5 for auxins and a two component system (TCSv2) for CKs have opened a window into the molecular mechanisms by which such interaction occurs during biosynthesis, transport and signaling.

14In recent years there has been a significant increase in the knowledge of the signal(s) that gives rise to the SE process, but it is still unknown if auxins are the primary signal that initiates the changes in the genetic program that leads to the production of somatic embryos. In C. canephora, it has been shown that polar transport of the IAA is needed for the formation of the apical-basal axis. It has also been reported that CKs are essential to maintaining basal levels of auxin biosynthesis during root and shoot development, suggesting that there is a homeostatic regulatory network to support adequate concentrations between auxins and CKs in the development of the plant. It is possible that a similar system is operating during the induction of SE. However, this must be tested.

15The SE process implies the integration of endogenous signals and gene reprogramming, which unchains the signal that initiates the embryogenic process. The use of exogenous auxins, either alone or in combination with other PGRs or stress, induces the expression of different genes, which modify the genetic program of the somatic cells and regulate the transition to each of the stages during the development of SE. Most of these genes belong to one of these four categories: transcription factors (TFs), proteins that act in the cell cycle, biosynthesis of PGR, mainly auxins, as well as proteins involved in the signaling pathway.

16It is generally accepted that the SE process involves three phases: the induction of SE, the formation of the meristematic centers, and the development of the somatic embryo. Each stage comprises the interaction of multiple factors, e.g., external signals, changes in the endogenous concentrations of different PGRs, and the expression of numerous genes. Molecular studies of the induction of SE are challenging since it is difficult to identify the cells that will become new somatic embryos. However, it is possible to carry out bioinformatics analysis from transcriptomic studies gain a better picture of the candidate genes involved in the initiation of the process.

17Production of the signal that leads to the changes in the genetic program requires the participation of several metabolic pathways. However, there is a consensus that auxins play a critical role in the SE process. It is known that auxin plays a crucial role in the formation of embryo patterns in angiosperms and in gymnosperms. During the induction of SE in C. canephora, there is an increase in the content of endogenous IAA and in the expression of the genes that code for the enzyme tryptophan aminotransferase (TRYPTOPHAN AMINOTRANSFERASE OF ARABIDOPSIS 1; CcTAA1), and for the enzyme flavin mono-oxygenase (YUCCA; CcYUC1 and CcYUC3). Both are involved in the biosynthesis of IAA.

18The response of the explant is not confined to the increase in the IAA levels. Differential gene expression can modulate the embryogenic capacity of cells, and the number of genes turned off in somatic cells to allow for the change from a somatic to an embryogenic state is higher than the number of genes that are turned on. In the SE of Arabidopsis, the modulation of several AUXIN RESPONSE FACTORS (ARF) transcripts suggests the extensive participation of auxin signaling during the process. Almost half of the 23 ARF genes are transcribed during SE in Arabidopsis; six of them are upregulated and five are down-regulated. Other members of the auxin signal transduction pathway, like the putative Aux/IAA gene from Elaeis guineensis, EgIAA9, or cotton, are also involved in the induction of SE. An extensive analysis of gene expression during the induction of SE in cotton shows that more than 80 genes related to the metabolism of auxins are differentially expressed.

19Somatic embryogenesis is a multifactorial event, which is the result of a series of physiological, biochemical and molecular changes taking place in plant cells. SE requires embryogenic competence through dedifferentiation, chromatin remodeling, programming of gene expression, and stress events mentioned above. In general, the SE induction includes a multitude of parallel signals that involve alterations in the levels of endogenous PGR and stress factors.

20Different studies support the theory that the first stages of SE are characterized by the induction of numerous genes related to stress such as those discussed later on this review. Recent evidence in potato, Pinus sylvestris, Picea asperata, Oldenlandia umbellata, and Cyathea delgadii has revealed that the presence of different types of stress plays an essential role in the induction of SE. The main stress for cells during the induction of SE is the presence of high auxin concentration in the culture medium. Other stresses used for the induction of SE are extreme pH, heat-shock exposure or treatment with various chemical substances.

21Usually, the combination of physical stress with high auxin concentration in the culture medium improves the embryogenic response. This effect was observed in Cattleya maxim where the effect in the SE induction was evaluated using a combination of salt (0.3 M NaCl) or osmotic stress (sorbitol 0.4 M), and the culture in a medium supplemented with 2,4-D (0.45 μM) significantly increases the percentage of protocorms with embryogenic calli. In some angiosperms such as Panax ginseng, the treatment of somatic embryos with abscisic acid (ABA) and polyethylene glycol (PEG) at a concentration of 20 μM and 3.75%, respectively, improve both the maturation and regeneration of somatic embryos compared to the untreated. However, in gymnosperms, the combined application of ABA and PEG has been shown to be necessary to stimulate the maturation and functional development of somatic embryos. For example, in Pinus sylvestris, embryo production is commonly induced by eliminating auxin from the culture medium, ABA addition and subsequently a PEG drying step. In P. strobus, variable amounts of water at the beginning and during the cultivation phase influences the maturation response of the embryos. Meanwhile, changes in water availability either by solutes or physical restriction can affect the maturation response in some conifers. Other types of stress like heat-shock induce the SE in Gladiolus hybridus. In cotton, several of the genes expressed during the induction of SE are related to the homeostasis of auxins and ethylene, as well as several related-stress TFs.

22There is very little current information on whether the genes involved in the induction of SE work independently or in a network-like structure. However, the analysis of the interaction among different clusters of genes shows that they can act in parallel or in sequence. The use of transcriptomics has provided valuable. Indicates that the genes expressed during the induction of SE are divided into the categories of stress-related genes, PGR-related genes, and TFs.

23The changes in the genetic program of the cells that lead to the induction of SE require the regulation of several genes. In both angiosperms and gymnosperms, little is known about gene expression, the early stages of embryogenesis, which is crucial for the later development of the embryo. For example, it has been reported that in conifers such as Araucaria angustifolia that the expression patterns of AaSERK1 during SE are very similar to SERK1 homologs of angiosperms. These changes require the substantial participation of TFs. Plant genomes contain a large number (6–10%) of TFs-coding genes. Some of these TFs are shared among a variety of plant species (Supplementary Table S1). Among the TFs that have been found during the induction of SE in different species are ABAINSENSITIVE 3 (ABI3), AGAMOUS LIKE (AGL), BABY BOOM (BBM), CUP SHAPED COTYLEDONS (CUC), FUSCA3 (FUS3), LEAFY COTYLEDON (LEC), LEAFY COTYLEDON LIKE (LIL), SOMATIC EMBRYOGENESIS RECEPTOR-LIKE KINASE1 (SERK1), RWP-RK DOMAIN-CONTAINING 4 (RKD4)/GROUNDED (GRD). VIVIPAROUS1 (VP1), and WUSCHEL (WUS). In conifers, several homologs of important genes that participate during ES have been found, such as SERK1, LEC1, and WOX2, but it is still unknown whether they present patterns and expression functions similar to angiosperms. Several of these genes are also expressed during the formation of zygotic embryos. The application of auxins or their analogs, like 2, 4-D, enhances the expression of several TFs, such as BBM, WUS, and VP1 during the induction of SE.

24In some cases, like the SE induced in wounded tissues, there is a signal that occurs before to the expression of the TFs listed in the last paragraph. The expression of WOUND INDUCED DEDIFFERENTIATION1 (WIND1) TF, from the AP2/ERF family, is required before the expression of LEAFY COTYLEDON2 (LEC2) takes place. The expression of some TFs is specific to particular species; however, several others are expressed in all the systems of induction of SE studied. The roles of these TFs in the signaling process are discussed below.

25Among the different genes that increase their expression during the induction of SE, SERK is the most relevant. This family of TFs is involved in a range of developmental processes that include differentiation/transdifferentiation and cellular totipotency.

26The first SERK gene was identified in D. carota. It was detected in embryogenic cultures in the early days of culture in the presence of 2, 4-D. This gene is expressed in cells that develop in somatic embryos until the globular stage, just before the transition from the differentiation state to the development state. The expression of SERK increases several times in the embryogenic cells of A. thaliana, Citrus unshiu, Dactylis glomerata, G. hirsutum, Helianthus annuus, Medicago truncatula, Solanum tuberosum, Vitis vinifera, Cocos nucifera, Oryza sativa, Theobroma cacao, Triticum aestivum, Zea mays, Cyrtochilum loxense, and A. angustifolia.

27The evidence of the participation of SERK in the induction of SE has emerged from the analysis of gene expression. For example, SERK1 is highly expressed during the formation of embryogenic cells in in vitro culture of A. thaliana and in all of the cells of the developing embryo during early SE, up until the heart stage of the somatic embryo. After this stage, the expression of SERK1 is no longer detectable in the embryo. However, in seedlings that over-expressed SERK1, the mRNA exhibited a 300–400% increase in the efficiency of the initiation of SE. These results suggest that an increase in the expression levels of SERK1 confers embryogenic competence to cells in culture. In O. sativa, SERK2 is expressed almost three times more in the embryogenic callus and maturation stage than in the non-embryogenic callus. These results suggest that different members of the SERK family have unique functions. Similar results have been found in T. aestivum. In this plant, members of the SERK family are expressed differentially in response to different PGR sensitivities; i.e., SERK2 and SERK3 elicit auxin-specific responses while SERK1 and SERK5 may be mediated by the signaling pathway of brassinosteroids.

28In addition to auxins, other factors modified the expression of SERK. In M. truncatula, the expression of SERK1 is stimulated by the presence of auxin, but not by CKs. However, when the CKs are co-administered with auxin, the level of expression of SERK1 increases synergistically compared to the up-regulation of auxin alone. In response to a higher level of expression of SERK, the number of embryogenic calluses increase as well as the formation of somatic embryos.

29Another important participant in the regulation of SE and plant embryo development is the LEC family of TFs. LEC1 has an essential role in ZE and has been suggested to control diverse processes in seed development, including embryo morphogenesis, maturation phases, germination, and early and late embryogenesis; it also appears to allow the formation of the embryo by establishing an embryonic environment. LEC1 is also involved in photosynthesis and chloroplast biogenesis early in seed development, and seed maturation late in the development of zygotic embryos. This gene network regulated by LEC1 has been conserved in dicotyledonous plants that diverged tens of millions of years ago.

30LEC1 and LEC2 were the first TFs shown to induce SE when ectopically expressed in seedlings. The auxin-dependent upregulation of LEC2 has been associated with the induction of SE, whereas LEC2 expression was markedly lower in non-embryogenic callus of A. thaliana, suggesting that LEC2 mediates the increase in the endogenous auxins observed during the induction of SE. Similar results were found in T. cacao, where LEC2 is highly expressed in the embryogenic callus and its overexpression in cotyledon explants increased the embryogenic response. The ectopic overexpression of LEC2 from Ricinus communis in A. thaliana induces the expression of TFs such as LEC1, L1L, FUS3, ABI3, and WRINKELED1 (WRI1). Also, the expression of the fatty acid elongase 1 (FAE1) and, in consequence, an accumulation of triacylglycerols, especially those containing the seed-specific fatty acid, eicosenoic acid (20:1 Δ11), in vegetative tissues was observed.

31The establishment of the shoot apical meristem (SAM) is essential for SE and for shoot regeneration. These processes require the expression of WUS, which encodes a bifunctional homeodomain TF. WUS contains a highly conserved homeobox domain, and at the conserved C terminal region it has three functional domains: an acidic domain, a WUS-box (TLPLFPMH), and an EAR-like motif. A very important characteristic of WUS is its ability to move from one tissue to another. It can move from its biosynthesis site, the central zone (CZ), into the daughter cells in the peripheral zone, where it activates the transcription of CLAVATA3 (CVL3), a negative regulator. CLV3 moves into the extracellular space and binds to CLV1, which in turn inhibits the transcription of WUS. This WUS-CLV feedback system establishment maintains the stem cell pool and the development of SAM. Therefore, WUS has been proposed to be essential for SE and in vitro shoot regeneration.

32WUSCHEL, like LEC2, responds to the presence of auxins. Auxins trigger a signaling cascade that initiates the vegetative-to-embryogenic transition, and this transition is mediated by WUS. The gradient of auxins that is detected during the pre-treatment of C. canephora plantlets and later during the initial phases of SE correlates with the induced WUS expression during SE in A. thaliana.

33It has been observed that WUS-related genes are up-regulated during SE in different species, such as Ocotea catharinensis, M. truncatula, G. hirsutum, and C. canephora. In C. canephora, overexpression of WUS enhances SE in heterologous systems, increasing the somatic embryo production by 400%. In G. hirsutum, the ectopic expression of AtWUS promotes the proliferation and differentiation of transgenic callus and positively regulates LEC1, LEC2, and FUS3. WUS overexpression enhances the induction of SE and can improve regeneration in cotton, and its overexpression in A. thaliana roots, leaf petioles, stems, or leaves induces the formation of somatic embryos.

34Another key regulator of plant cell totipotency is BBM. BBM can induce embryogenesis in differentiated cells and could be a vital factor in plant embryogenesis development. BBM triggers a set of genes like LEC1 and LEC2, as well as ABI3 and the FUS3 network, which together activate SE. The induction of SE by BBM is a dose-dependent mechanism and regulates the transcription of significant embryo identity genes.

35The BBM family encodes APETALA 2/ETHYLENE RESPONSE FACTOR (AP2/ERF) DNA-binding type TFs identified in the gymnosperms, angiosperms, algae, and mosses, these TFs act as a network regulation in response to biotic and abiotic stress. The AP2/ERF domain can bind to a GCC box, a DNA sequence involved in the ethylene response. AP2/ERF are divided according to the number of AP2 domains that they contain, which are classified into subfamilies as the Dehydration-responsive 427 element-binding (DREB), ERF, AP2, and RELATED TO ABI3/VP1 (RAV) genes. Because RAV genes include another DNA-binding domain, B3, RAV genes are sometimes treated as a third group in the AP2/ERF family. The distinct feature of the BBM and BBM-like proteins is the presence of a conserved bbm-1 motif (GLSMIKTW) that is absent in other proteins of the euANT lineage. BBM activated the expression of a broad set of genes encoding proteins with potential roles in transcription, cellular signaling, cell wall biosynthesis and targeted protein turnover, such as the ACTIN DEPOLYMERIZING FACTOR9 (ADF9).

36In A. thaliana and B. napus, BBM changes its spatial-temporal expression in the early stages of embryogenesis. Some reports show that BBM is expressed in the heart state of an embryo and root development and enhances the proliferation of somatic embryos. This response is also produced by ectopic expression of BBM, which changes from vegetative to embryonic growth and induces spontaneous SE in these two species. The heterologous expression of BBM from A. thaliana and B. napus in Nicotiana tabacum produced an increase in the regeneration capability. In Capsicum annum, both LEC1 and BBM are expressed and show high levels of expression in the different phases of development of the somatic embryo.

37On the other hand, it is worth highlighting that BBM can show differential expression depending on the species and the embryogenic protocol. In a study using two species of the genus Coffea, it was found that while in C. arabica a BBM-like gene showed a twofold change in expression in embryogenic cell suspension in comparison to embryogenic calli, in C. canephora BBM1 expression was only observed after SE induction. It has been found that the BBM gene is expressed at higher levels during SE in comparison to ZE in T. cacao, and its overexpression in A. thaliana and T. cacao led to phenotypes associated with SE that did not require exogenous hormones. However, BBM overexpression can inhibit the subsequent development of the somatic embryos in T. cacao, while the BBM overexpression in Populus tomentosa induced SE.

38Somatic embryogenesis signaling is a very complex process where several molecular players are involved; it would be tedious to list them all. However, there are two other major factors that need to be mentioned. One is the intervention of 14-3-3 proteins, which participate in several processes such as the development of the seeds and during the induction of SE in Carica papaya. The other factor actively involved during the SE induction, process, and development is epigenetic.

3914-3-3 adaptor proteins are a group of proteins involved in the signal transduction pathway that is shared by several PGRs involved in SE induction. These proteins are highly conserved phosphoserine-/phosphothreonine-binding proteins, discovered in the brain of mammals in 1967, with a subunit mass of 30 kDa.

40In plants the number of members of these proteins is variable (Figure 2). There are 13 14-3-3 adaptor proteins in Arabidopsis, six in cotton, 17 in tobacco, ten in tomato, five in barley, and eight in rice.

41The use of proteomics techniques has illuminated the changes in hundreds of proteins, including the family 14-3-3, during the induction of SE. Some 14-3-3 proteins are abundant in the embryogenic tissues of Cyclamen persicum, and Larix principis. In oak, these proteins are more abundant in proliferating embryos than in mature embryos.

42An excellent example that shows the role of 14-3-3 proteins in the induction of SE is protein phosphatase 2A (PP2A). This enzyme consists of a catalytic subunit and a regulatory A subunit together with a third variable B subunit. The B subunit is the component that determines the substrate specificity and subcellular localization of PP2As. PP2A is a complex enzyme. In A. thaliana, there are 25 genes involved in the transcription of PP2A three subunits. The catalytic subunit (PP2Ac) is coded by five genes, three other genes encoding A subunits and seventeen different genes encoding B subunits. The subunit A is essential for auxin transport, while the 65 kDa regulatory subunit of PP2A has regulatory functions. The subunit A has been associated with the SE process. There is a noticeable increase in phosphorylation of specific proteins in embryogenic cultures compared to the non-embryogenic cells of C. persicum, which has been correlated with higher levels of PP2A and a 14-3-3-like protein. Other components of the signal transduction cascade, such as G proteins and calreticulin, increased during cyclamen SE. It has been suggested that the increase in the regulatory subunit of PP2A and 14-3-3 proteins during the induction of SE is related to the stress conditions produced by the in vitro culturing of C. persicum and L. principis embryogenic cultures.

43In recent years, epigenetic mechanisms during chromatin remodeling have emerged as critical factors in SE. Epigenetic modifications are an essential part of the signaling pathway that leads to changes in the genetic program of the cells and the development of somatic embryos. There is evidence that shows that changes in the chromatin are able to control totipotency in plant cells. The level to which chromatin reprogramming is required before SE induction depends on several factors, such as origin of the explant, the culture medium, the genetic background of the mother plant, and especially the amount of PGR used.

44DNA methylation is important for somatic embryo development. In general, higher global DNA methylation has been found in non-embryogenic cultures of Pinus radiata, P. nigra, Rosa x hybrid, and Eleutherococcus senticosus, while low global DNA methylation has been found in embryogenic cultures of several plants. In Quercus alba DNA is demethylation during the induction of SE, as well as during the generation of pro-embryogenic mass, but it gradually increases as the embryo is developing. Similar results were observed during the SE of C. canephora, where the proembryogenic mass had lower DNA methylation, while the maturation of the embryos was marked by a gradual increase in the global levels of methylation.

45In A. thaliana it was found that both de novo DNA methylation and maintenance of it are required for the regulation of SE, and similar results were found in Picea abies. Changes in the global DNA methylation pattern during long-term subcultures could lead to the loss of the embryonic potential of proembryogenic masses.

46In order to prove that in fact DNA methylation is strongly related to SE, pharmacological experiments have been conducted in several plant species. The application of 5-azacitidine (5-AzaC; a demethylating agent) decreased the levels of global DNA methylation in A. thaliana and inhibited the induction of SE. Similar results have been found in M. truncatula, D. carota, and C. canephora. Furthermore, LEC1, LEC2, and BBM genes were up-regulated in the drm1drm2 and drm1drm2cmt3 mutants, an upregulation that was related to an improvement in the SE response. In T. cacao, DNA methylation increased during the induction of SE, and treatment with 5-AzaC led to the recovery of SE potential in aged cultures. 5-AzaC is not the only drug used to disrupt epigenetic modifications; trichostatin A (TSA), the function of which is inhibiting histone deacetylases (HDACs), has a positive effect on gene expression. The inhibition of HDACs has also led to an increase in the number of haploid embryos produced by heat stress in B. napus. In fact, the treatment with TSA of germinating spruce somatic embryos preserves their embryogenic nature. In the double mutant hda6/hda19, the upregulation of LEC1, FUS3, and ABI3 genes was evident in germinating Arabidopsis seeds. These double mutants also led to the production of somatic embryos in the leaves of Arabidopsis.

47Histones’ posttranslational modifications have been implicated in the formation of somatic embryos. Histone deacetylation may also play a role in the reprogramming of cells in the early stages of SE, since the levels of histone acetylation and the activity of HDACs change in response to the presence of exogenous PGR during the induction of SE.

48There are several tissue-specific events involving H3K27me3. The loss of this mark upregulates the auxin pathway and its increase leads to the repression of leaf identity. Polycomb repressive complex 2 (PRC2) is involved in the methylation of lysine 27 in histone H3. Double mutants of the PRC2 gene, which functions as a histone methyltransferase, CLF and SWN or VERNALIZATION 2 (VRN2) and EMBRYONIC FLOWER2 (EMF2) form callus on the shoot apex, lead to indirect somatic embryo formation and ectopic roots. A PRC2 mutant root hairs fail to maintain their differentiated state and form unorganized cell masses and eventually somatic embryos from callus. The effect of silencing genes of the PCR2 family in inducing SE depends on the explant. In tissues where PCR2 is scarcely active, the production of somatic embryos is efficient; however, in the tissues where it is highly expressed somatic embryos do not form.

49Since the 1950s, the research on the SE process has gone from empirical approaches to a more methodical investigation leading to the production of somatic embryos. We are well on the way to understanding the role of auxins and other PGRs, as well as stress, on the induction of SE. We now have a set of genes that, in some cases, can be used as markers of the initiation of SE. However, the signal pathway from the initial signal to the first steps of the development of the somatic embryo remains practically unknown.

50Scientists have just begun to understand the complex network of interactions among a set of TFs, the endogenous concentrations of auxins, CKs, ABA, ethylene and salicylic acid, their transport and receptors, and the origin of the explant that lead to the establishment of a somatic embryo (Figure 3 and Supplementary Table S2).

51Current scientific knowledge lets us hypothesize that the initial signal, stress or the signals produced by the PGRs induce a change in the endogenous concentration of several PGRs, especially auxins and CKs. The differences in the relationship between the auxins and CKs lead to the expression of TFs and ARF, which in turn modify the cell wall, a vital component in the cell differentiation process. Once the cell(s) are settled into the SE pathway, the expression of TFs, such as BBM, SERC, and LEC, leads to downstream changes in the endogenous content of different compounds and produces a cascade of events, such as chromatin remolding, that drives the induction of SE. However, there are still many questions to answer to understand how the life of a somatic embryo begins. The roles of ethylene, salicylic acid, the organization of the cytoskeleton, brassinosteroids, and other compounds remain to be elucidated.

52The overexpression of genes such as WUS, BBM, and LEC has been used to induce SE in different plant species. This approach has been instrumental in understanding the role of different genes during the induction of SE; however, under certain conditions, the overexpression also inhibits the induction of SE. This means that under the present state of the art, every gene and every plant species must be tested, before all of the pieces of the puzzle are in place.

53Increasing knowledge of the induction of SE and of the development of somatic embryos will lead to the development of multiple biotechnological applications and new opportunities for the understanding of the fundamental aspects of SE. In particular, the alteration in the methylation or acetylation profile of DNA and/or histones by genome-editing techniques holds great promise to increase the production and to improve the quality of crops of agronomical importance.

54VL-V developed the idea. All authors drafted the manuscript.

55The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.