REVIEW
New insights into plant somatic embryogenesis: an epigenetic view
Vijay Kumar1 • Johannes Van Staden1
Received: 8 March 2017 / Revised: 17 May 2017 / Accepted: 23 July 2017 / Published online: 2 August 2017
� Franciszek Gorski Institute of Plant Physiology, Polish Academy of Sciences, Krakow 2017
Abstract Somatic embryogenesis plays a significant role
in plant regeneration and requires complex cellular,
molecular, and biochemical processes for embryo initiation
and development associated with plant epigenetics. Epi-
genetic regulation encompasses many sensitive events and
plays a vital role in gene expression through DNA
methylation, chromatin remodelling, and small RNAs.
Recently, regulation of epigenetic mechanisms has been
recognized as the most promising occurrences during
somatic embryogenesis in plants. A few reports demon-
strated that the level of DNA methylation can alter in
embryogenic cells under in vitro environments. Changes or
modification in DNA methylation patterns is linked with
regulatory mechanisms of various candidate marker genes,
involved in the initiation and development of somatic
embryogenesis in plants. This review summarizes the
current scenario of the role of epigenetic mechanisms as
candidate markers during somatic embryogenesis. It also
delivers a comprehensive and systematic analysis of more
recent discoveries on expression of embryogenic-regulat-
ing genes during somatic embryogenesis, epigenetic vari-
ation. Biotechnological applications of epigenetics as well
as new opportunities or future perspectives in the devel-
opment of somatic embryogenesis studies are covered.
Further research on such strategies may serve as exciting
interaction models of epigenetic regulation in plant
embryogenesis and designing novel approaches for plant
productivity and crop improvement at molecular levels.
Keywords Chromatin remodelling � DNA methylation �Epigenetics � Somatic embryogenesis
Abbreviations
5-azaC 5-Azacytidine
AGL15 Agamous-Like15
BBM1 Baby Boom1
CMT Chromomethylase
CRED-RA Coupling of restriction enzyme and aleatory
amplification
CLF Curly leaf
2,4-D 2,4-Dichlorophenoxyacetic acid
DCMtases DNA cytosine methyltransferases
DCL1 Dicer-like 1
DRM Domain rearranged methyltransferase
DSE Direct somatic embryogenesis
GLPs Germins and germin-like proteins
HPCE High-performance capillary electrophoresis
HPLC High-performance liquid chromatography
IAA30 Indole acetic acid inducible 30
ISE Indirect somatic embryogenesis
LEC Leafy cotyledon
5-mC 5-Methylcytosine
MET Methyltransferase
MSAP Methylation-sensitive amplification
polymorphism
PGRs Plant growth regulators
PRC1 Protein regulator of cytokinesis1
SAH S-Adenosyl-L-homocysteine
SAM S-Adenosyl-L-methionine
SE Somatic embryogenesis
SERK Somatic embryogenesis receptor kinase
Communicated by J. Van Huylenbroeck.
& Johannes Van Staden
1 Research Centre for Plant Growth and Development, School
of Life Sciences, University of KwaZulu-Natal
Pietermaritzburg, Private Bag X01, Scottsville 3209, South
Africa
123
Acta Physiol Plant (2017) 39:194
DOI 10.1007/s11738-017-2487-5
WIND3 Wound-induced dedifferentiation3
WOX4 Wuschel-related HOMEOBOX4
WUS Wuschel
ZE Zygotic embryogenesis
Introduction
In vitro plant regeneration is often achieved via organo-
genesis or embryogenesis. Somatic embryogenesis (SE) is
a unique process in the life cycle of most plants and has
become an essential tool in plant biotechnology mainly for
mass propagation and crop improvement for commercial
application. During developmental processes, competent
somatic cells undergo modification through a set of chan-
ges; cellular, morphological, molecular, and biochemical to
convert into embryogenic cells. In the somatic embryoge-
nesis pathway, zygotic embryo development represents
different developmental stages such as globular, juvenile,
and coleoptile-shaped in monocotyledonous plants
(Mordhorst et al. 1997); globular, heart, torpedo, and
cotyledonary-shaped in dicotyledonous plants and globu-
lar, early, and late cotyledonary-shaped present in conifers
(Yang and Zhang 2010). Somatic embryogenesis is a
powerful alternative to the conventional mass propagation
and a novel model system for crop improvement with the
traditional agricultural methods (Loyola-Vargas et al.
2008). The induction of SE is a complex multi-factorial
system involving endogenous hormones, and is mainly
stimulated by exogenous PGRs (Jimenez 2005). The
mechanisms inducing embryogenesis are complex; SE
relies on a complicated series of interactions between dif-
ferent PGRs, mostly auxins alone or in combination with
different cytokinins, during the initial proembryogenic
phases. Ethylene, abscisic acid, and gibberellic acid are
involved during maturation and production of somatic
embryos (De-la-Pena et al. 2015; Kumar et al.
2015a, 2016, 2017). A somatic embryogenesis pathway
occurred either by indirect somatic embryogenesis (ISE) or
direct somatic embryogenesis (DSE) (Fig. 1). As shown in
Fig. 1, stress and exogenous hormones are involved in the
expression of various genes such as Auxin Response Factor
(ARF7, ARF19) and Protein Regulator of Cytokinesis
(PRC1). After the cell achieves dedifferentiation potential,
Leafy Cotyledon (LEC1 and LEC2) genes are expressed
and increase the endogenous auxin level, which conse-
quently upregulates the expression of Curly Leaf (CLF),
Wuschel (WUS), and Somatic Embryogenesis Receptor
Kinase (SERK). At this step, few physiological changes
such as auxin signalling and chromatin remodelling could
result in the expression of totipotency. Therefore, the
embryogenic cells develop into somatic embryos. The
molecular mechanism controlling the somatic
embryogenesis process requires further study. A molecular
mechanism of somatic embryogenesis has been made in
Arabidopsis (Fig. 2). It is difficult to understand the
molecular mechanisms regulating embryogenesis in all
types of plant species. However, identification of several
genes in Arabidopsis will help to understand the molecular
network in somatic embryogenesis. As shown in Fig. 2,
several regulatory genes such as Polycomb repressive
complex1/2 (PRC1/2) and PICKLE (PKL) subsequently
induce the LEC1, LEC2, and FUSCA3 (FUS3) and the
transcription factor AGAMOUS-LIKE15 (AGL15) during
somatic embryogenesis which control several downstream
physiological processes to promote embryonic compe-
tence. LEC1 induces the YUC10 gene and LEC2 activates
the YUC2 and YUC4 gene, which encodes an auxin
biosynthesis enzyme (Junker et al. 2012; Stone et al. 2008).
Furthermore, LEC2 and AGL15 expressed the negative
regulator of auxin signalling, an INDOLE ACETIC ACID
INDUCIBLE30 (IAA30), which modulate the auxin-medi-
ated signalling during embryogenesis (Braybrook et al.
2006; Zheng et al. 2009). In addition, AGL15 positively
regulates Gibberellin (GA) degrading enzyme GA2ox6, and
negatively regulates the biosynthesis gene GA3ox2,
resulting in the reduced endogenous GA level (Wang et al.
2004; Zheng et al. 2009; Guan et al. 2016; Ikeuchi et al.
2016). The FUS3 repressed GA3ox1 and GA3ox2, resulting
downregulating GA biosynthesis as transcriptional regula-
tion mechanisms yet not clear (Curaba et al. 2004; Gaz-
zarrini et al. 2004; Guan et al. 2016; Ikeuchi et al. 2016).
Under in vitro conditions, different stages of embryos
develop from the explant/tissue directly or through callus
formation on the surface of seedling tissues. According to
Willemsen and Scheres (2004), embryogenic competent
cells are present in DSE, whereas an essential gene mod-
ification/reprogramming is required for embryogenic callus
induction and differentiation before development of
somatic embryos in ISE (Williams and Maheswaran 1986;
Yeung 1995). Both types of calli (non-embryogenic and
embryogenic) are present during ISE. In general, it is quite
easy to differentiate between non-embryogenic and
embryogenic callus because of their morphology and col-
our. Embryogenic callus has a smooth surface with nodular
structures, somatic embryos produced by embryogenic
cells are isodiametric in shape and small in structure, while
non-embryogenic callus is friable with rough structures and
is translucent (Jimenez and Bangerth 2001; Yang and
Zhang 2010). Since the very first reports by Stewards et al.
(1958) and Reinert (1959) on somatic embryo production
in carrot cell suspensions, the potential for the induction
and formation of somatic embryogenesis has been devel-
oped for many dicotyledon, monocotyledonous, and gym-
nosperms plants (Uddin 1993; Stasolla et al. 2004; Mathieu
et al. 2006; Quiroz-Figueroa et al. 2006). Somatic
194 Page 2 of 17 Acta Physiol Plant (2017) 39:194
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embryogenesis inducing mechanisms are complicated and
quite similar amongst different plant species. To induce
somatic embryogenesis, cell division, cell dedifferentia-
tion, and changes in the physiology and metabolism of the
cells are crucial factors (Elhiti et al. 2013; Mahdavi-Dar-
vari et al. 2015). Due to the significant role of epigenetic
regulation, the present review provides current under-
standing of different epigenetic mechanisms that regulate
plant embryogenesis. This review also focuses on expres-
sion of embryogenic-regulating genes during somatic
embryogenesis, epigenetic variation, and biotechnological
applications of epigenetics, as well as new opportunities or
future perspectives in the development and improvement of
somatic embryogenesis studies. Therefore, the knowledge
about the epigenetic mechanisms during somatic embryo-
genesis could help to enhance the embryogenic capacity of
different plant species and also improve new approaches
for plant breeding and crop improvement.
Epigenetic regulation in somatic embryogenesis
The term ‘‘epigenetics’’ refers to genetic changes in gene
expression that are independent of DNA sequence variation
(Haig 2004; Berger et al. 2009; Zhang and Hsieh 2013).
Epigenetic mechanisms are extremely dynamic actions that
control gene expression. In recent years, it has developed
as critical factors during somatic embryogenesis (Nic-Can
and De-la-Pena 2014). Through DNA modification and
histone proteins in chromatin, these epigenetic mechanism
control gene programming. In plant cells, epigenetic
mechanisms are engineered by methylation of DNA,
chromatin remodelling, and microRNA-mediated regula-
tion (Miguel and Marum 2011; Neelakandan and Wang
2012; Mahdavi-Darvari et al. 2015; Ikeuchi et al. 2016).
For the successful achievement of somatic embryogenesis,
DNA methylation remains crucial.
DNA methylation
DNA methylation is an essential epigenetic mechanism
involved in different biological processes. It is a crucial
factor of the epigenome that regulates and maintains gene
expression programs (Milutinovic et al. 2003). Furthermore,
it plays a significant role in the differentiation and growth
regulation in plants through RNA-directed DNA methyla-
tion. It is a type of epigenetic marking that modulates
Fig. 1 Two different hypothesized pathways of somatic embryoge-
nesis. The somatic embryogenesis pathway can be induced in
differentiated cells directly or indirectly through callus/proembryo-
genic cell mass. Endogenous or exogenous (stress or plant growth
regulators or their combinations) signals can result leading to
dedifferentiation in competent cells followed by direct somatic
embryogenesis and callus tissue differentiation
Acta Physiol Plant (2017) 39:194 Page 3 of 17 194
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transcriptional activity of specific DNA sequences. DNA
methylation refers to the methyl group addition at the
5-carbon of cytosine base positions (Fig. 3). In plants, DNA
methylation occurs in the context of CG and CHG bases and
CHH nucleotide sequences (H = A, T or C). DNA methy-
lation is catalysed by a set of enzymes named DNA cytosine
methyltransferases (DCMTases), domain rearranged
methyltransferase (DRM), methyltransferase (MET), and
chromomethylase (CMT). DRM1 and DRM2 are responsi-
ble for the de novo methylation by miRNA-directed path-
way; however, maintenance of DNA methylation is
performed by MET1 and CMT1 (Cao and Jacobsen 2002;
Feher 2015). A wide range of factors affect embryogenesis in
plants (Elmeer 2013; Joshi et al. 2013); however, DNA
methylation plays a vital role in cellular dedifferentiation, re-
differentiation, and somatic embryogenesis in plants and
also controls plant growth and development (Fig. 4) (He
et al. 2011; Nic-can et al. 2013). Changes in DNA methy-
lation in dedifferentiating Arabidopsis leaf protoplasts have
been reported (Avivi et al. 2004).
In Eleutherococcus senticosus somatic embryogenesis,
Chakrabarty et al. (2003) assessed the extent and pattern of
cytosine methylation using direct determination of
5-methyl-deoxycytidine (5mdC) amounts in genomic DNA
by quantification of nucleosides and methylation-sensitive
amplification polymorphism (MSAP) techniques and
HPLC separation.
HPLC analysis on genomic DNA from both embryo-
genic and non-embryogenic lines showed different global
DNA methylation level. The authors reported a low level
of global DNA methylation in E. senticosus embryogenic
callus when compared to non-embryogenic.
Fig. 2 Schematic molecular
model showing regulatory
interactions of somatic
embryogenesis in Arabidopsis.
Regulatory genes like PRC1/2
and PKL subsequently induces
the expression of LEC1, LEC2,
and FUS3 which together with
AGL15 modulate the
endogenous levels of auxin
signalling and GA to promote
somatic embryogenesis. Arrows
with a solid line indicate direct
transcriptional regulation by
molecular evidence and arrows
with dotted line indicates
transcriptional regulation that
molecular mechanisms are not
clear
Fig. 3 Cytosine methylation. S-
Adenosyl-L-homocysteine
(SAH); S-adenosyl-L-
methionine (SAM)
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Similarly, by using the MSAP technique, DNA methy-
lation level was found up to 16.99% in non-embryogenic
callus, whereas embryogenic callus methylation was
11.20%. In Pinus nigra, low methylation levels were
observed in embryogenic cell lines (Noceda et al. 2009). In
carrot embryogenesis, the removal of auxin (2,4-D) causes
a loss of methylation content during embryo development.
However, methylation increases again during embryo
maturation (Lo Schiavo et al. 1989; Munksgaard et al.
1995). Cytosine methylation was promoted in carrot
somatic embryogenesis with increased 2,4-D concentra-
tion. Leljak-Levanic et al. (2004) investigated the alteration
in DNA methylation levels in Cucurbita pepo L. during
somatic embryogenesis. In the study, a significant level
was found in the early stage embryo and reduction was
found during embryo maturation in medium treated with
12.3 mM 5-azacytidine (5-azaC). The embryonic features
were maintained after 2 months when medium supple-
mented with 5-azaC, suggesting that embryogenesis could
be induced by stressful conditions and through changes in
methylation levels.
Similarly, in Medicago truncatula (Santos and Fev-
ereiro, 2002) and Acca sellowiana (Fraga et al. 2012),
AzaC is responsible for the reduction of DNA methylation
level in embryogenic cells. Treatment with 5-azaC caused
a loss of callus proliferation of the non-embryogenic line,
and decreased the rate of regeneration capacity in the
embryogenic line by reducing the production of somatic
embryos (Santos and Fevereiro 2002).
In chestnut (Castanea sativa), Viejo et al. (2010)
demonstrated DNA methylation implications during sexual
embryogenesis. Fertilized ovules experience DNA methy-
lation, whereas companion ovules increase their methyla-
tion level and induce degradation. Transient DNA
methylation after fertilization is needed for somatic
embryos maturation.
As a significant and widely used method, more precise
and efficient approaches need to be developed to identify
the specific regions of methylation as well as the total
content to clarify its potential role in somatic embryogen-
esis. The different approaches for the determination of
DNA methylation can be classified into different categories
such as global DNA methylation, genome-wide analysis,
regional DNA methylation, detection of specific methyla-
tion patterns, DNA methylation analysis by sequencing,
and individual CpG analysis (De-la-Pena et al. 2015).
Several tools for DNA methylation in different species are
listed in Table 1.
Chromatin remodelling
Chromatin is a genetic material composed of proteins and
DNA, in the nucleus of eukaryotes. It plays a central role to
reinforce the DNA macromolecule to allow mitosis and
facilitates gene expression and DNA replication. DNA is
tightly condensed by being covered around nuclear pro-
teins called histones, to form chromatin. These histones
helps in the arrangement of DNA into structures called
Fig. 4 Schematic model
showing embryogenic
competence by methylation of
DNA. After DNA methylation,
chromatin remodelling occurs in
somatic cell. Finally, somatic
cells was undergoing gene
reprogramming for expression
of totipotency and acquiring the
embryogenic competence
Acta Physiol Plant (2017) 39:194 Page 5 of 17 194
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nucleosomes by providing a base on which the DNA can be
covered. A nucleosome consists of a DNA sequence of
approximately 145 base pairs covered with histones (H2A,
H2B, H3, and H4) (Butler 1983; Verbsky and Richards
2001; Mahdavi-Darvari et al. 2015).
Chromatin remodelling is the reorganization of chro-
matin, allowing transcription factors or other DNA binding
proteins to access DNA and control gene expression. It
plays a crucial role in establishing gene expression patterns
and maintaining epigenetic regulation through successive
rounds of mitosis that takes place within a cell lineage
(Reyes 2006; Exner and Hennig 2008; Jarillo et al. 2009).
Many reports suggested that chromatin remodelling is
involved in the cell dedifferentiation, genome stability
maintenance, and plant development (Williams et al. 2003;
Avivi et al. 2004; Han et al. 2015; Zhang et al. 2015a, b). A
low level of DNA methylation and reduction in H3 lysine 9
dimethylation (H3K9me2) and H3K9me3 promotes the
gene expression associated with cell dedifferentiation
(Grafi et al. 2007; Bouyer et al. 2011). Recently, in Ara-
bidopsis, it was establish that H3K27me3 regulates the
expression of approx. 9006 genes (Lafos et al. 2011), and
H3K9me2 is involved actively in expression of genes in
dedifferentiated conditions (Grafi et al. 2007). According
to Nic-Can et al. (2013), loss in DNA methylation level and
decrease of H3K9me2 and H3K27me3 were observed
during Coffea canephora embryogenesis. Similarly, Grafi
et al. (2007) and Lafos et al. (2011) found that reduction in
DNA methylation level associated with decreased
H3K9me2 and H3K27me3 levels permits the triggering of
cell dedifferentiation by expressing the genes.
Transcription factor genes, BABY BOOM1 (BBM1) and
LEAFY COTYLEDON1 (LEC1), crucial for somatic
embryogenesis induction, and cell differentiation are epi-
genetically regulated by H3K27me3, while WUSCHEL-
RELATED HOMEOBOX4 (WOX4) is regulated by the
repressive mark H3K9me2 by Chromatin Immunoprecipi-
tation (ChIP) assays (Lafos et al. 2011; Nic-Can et al.
Table 1 DNA methylation during in vitro somatic embryogenesis in plants
Family Species DNA methylation detection method References
Myrtaceae Acca sellowiana HPLC/CRED-RA Fraga et al. (2012), Cristofolini et al. (2014)
Poaceae Bambusa balcooa MSAP Gillis et al. (2007)
Fagaceae Castanea sativa HPCE Viejo et al. (2010)
Rutaceae Citrus paradisi MSAP Hao et al. (2004)
Rubiaceae Coffea canephora HPLC Nic-Can et al. (2013)
Cucurbitaceae Cucurbita pepo CRED-RA Leljak-Levanic et al. (2004)
Apiaceae Daucus carota HPLC Palmgren et al. (1991)
Arecaceae Elaeis guineensis HPLC, SssI-MAA, MSAP Jaligot et al. (2004)
Araliaceae Eleutherococcus senticosus MSAP Chakrabarty et al. (2003)
Iridaceae Freesia hybrida MSAP Gao et al. (2010)
Gentianaceae Gentiana pannonica HPLC reversed phase Fiuk et al. (q2010)
Poaceae Hordeum brevisubulatum MSAP Li et al. (2007)
Poaceae Hordeum vulgare MSAP Bednarek et al. (2007)
Lauraceae Ocotea catharinensis MSAP Hanani et al. (2010)
Poaceae Oryza sativa MS-RFLP Brown et al. (1990)
Pinaceae Picea omorika MS-RAPD Levanic et al. (2009)
Pinaceae Pinus nigra HPCE Noceda et al. (2009)
Pinaceae Pinus pinaster HPCE/MSAP Klimaszewska et al. (2009)
Pinaceae Pinus pinaster HPCE Marum (2009)
Fagaceae Quercus suber HPCE Perez et al. (2015b)
Rosaceae Rosa hybrida L. MS-AFLP Xu et al. (2004)
Solanaceae Solanum tuberosum MS-AFLP Sharma et al. (2007)
Malvaceae Theobroma cacao MSAP Lopez et al. (2010)
Vitaceae Vitis vinifera MSAP Schellenbaum et al. (2008)
Poaceae Zea mays MS-RFLP Kaeppler and Phillips (1993)
CRED-RA coupling of restriction enzyme and aleatory amplification, HPCE high-performance capillary electrophoresis, HPLC high-perfor-
mance liquid chromatography, MS methylation-sensitive, MSAP methyl-sensitive amplification polymorphism, SssI-MAA SssI-methylase
accepting assay
194 Page 6 of 17 Acta Physiol Plant (2017) 39:194
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2013). BBM1 is necessary for embryogenic cell induction
and proliferation during embryogenesis (Boutilier et al.
2002; Kulinska-Lukaszek et al. 2012) whereas LEC1 is an
essential regulator gene for embryogenesis which helps to
induce somatic embryogenesis by expression (Lotan et al.
1998). Reports also suggest that WOX4 expression is
essential to stimulate procambium differentiation in Ara-
bidopsis and tomato (Ji et al. 2010; Suer et al. 2011).
Recent study have shown that POLYCOMB REPRESSIVE
COMPLEX2 (PRC2) mutants, a chromatin modifier,
reprogram, and develop embryo-like structures (Ikeuchi
et al. 2015). The PRC2 complex may directly bind to
histone H3 lysine 27 (H3K27me3) and maintains tran-
scriptional repression (Holec and Berger, 2012). The
WOUND INDUCED DEDIFFERENTIATION3 (WIND3, a
reprogramming regulator) and LEC2 (embryonic regulator)
repressed by PRC2 and contribute to cellular reprogram-
ming in PRC2 mutants.
MicroRNA-mediated regulation
MicroRNAs (miRNAs), are classified as small interfering
RNA (siRNA) or small RNAs (sRNAs), small non-coding
genes, *22 nucleotides long, originating from the single-
stranded RNA region of hairpin folded structure usually
transcribed by RNA polymerase II (Pol II) (Lee et al. 2002;
Bartel 2004; Jia et al. 2011). Mature miRNA is produced
predominantly by type III endoribonuclease Dicer-like 1
(DCL1) enzyme (Park et al. 2002; Liu et al. 2005; Sunkar
et al. 2005). miRNAs play crucial roles in epigenetic pro-
cesses and also have a profound effect in controlling gene
expression in plants. They regulate gene expression post-
transcriptionally during several metabolic and biological
events by binding to the 30-untranslated region of target
mRNAs to inhibit translation or facilitate mRNA degra-
dation. It also plays an essential role as important regula-
tors in plants at various developmental stages and
facilitates organ identity maintenance (Jones-Rhoades et al.
2006). The significant role of miRNAs in various metabolic
and biological processes in plants is known, including
zygotic embryogenesis (ZE) in which miRNAs were doc-
umented to be crucial for the proper patterning and mor-
phology of the somatic embryos (Willmann et al. 2011;
Seefried et al. 2014; Vashisht and Nodine 2014; Wojcik
and Gaj 2016.). Recently, a number of studies suggested
the essential regulatory roles for miRNAs during somatic
embryogenesis (Luo et al. 2006; Nodine and Bartel 2010;
Willmann et al. 2011; Li et al. 2012a, b; Shen et al.
2012, 2013; Lin and Lai 2013; Qiao and Xiang 2013; Yang
et al. 2013; Chavez-Hernandez et al. 2015; Su et al. 2015;
Zhang et al. 2015a, b). According to these reports, the
patterns of miRNA change between embryogenic and non-
embryogenic callus induction as well as during
differentiation in a plant. For example, a significant
expression of miR171, miR390, miR397, and miR398 has
been found in rice embryogenic callus (Chen et al. 2011;
Wu et al. 2011). miRNA167 controls somatic embryoge-
nesis in Arabidopsis through regulating its target genes
ARF6 and ARF8 (Su et al. 2015). These reports summarize
significant information regarding miRNA-mediated
embryogenesis. During in vitro regeneration from maize
embryogenic callus, improved expression levels of
miR156, miR159, miR164, miR168, miR397, miR398,
miR408, and miR528 were observed (Chavez-Hernandez
et al. 2015). During somatic embryogenesis of cotton, 25
novel and 36 known miRNA were identified using a high-
throughput sequencing approach (Yang et al. 2013).
Among the several identified miRNA, miR156 exhibited a
significant expression in the embryogenic cells. A similar
finding has been observed in rice (Luo et al. 2006; Chen
et al. 2011), valencia sweet orange (Wu et al. 2011), and
hybrid yellow poplar (Li et al. 2012a, b). A significant level
of miR156 targets on members of Squamosa Promoter
Binding Protein-Like (SPL) transcription factor was acti-
vated throughout early somatic embryogenesis and also
during in plant development (Rhoades et al. 2002; Nodine
and Bartel, 2010). Similarly, in valencia sweet orange the
accumulation of miR156 was observed during initiation of
embryogenic calli (Wu et al. 2011). In Arabidopsis, early
embryogenesis miRNAs has been found as a candidate
mark for the regulation of transcriptional factor genes such
as LEC2 and FUS3 (Willmann et al. 2011), while in cotton,
homeobox-related WOX genes are known to be regulated
by miRNAs (Yang et al. 2013).
Expression of embryogenic-regulating genes
during plant embryogenesis
Somatic embryogenesis is a unique model system in plants
which provides a valuable tool to enhance the genetic
improvements of different crop species at molecular level
(Chugh and Khurana 2002). A number of studies at the
molecular level suggest that a limited number of genes are
involved during somatic embryogenesis in plants (Schrader
et al. 1997; Hu et al. 2005; Ikeuchi et al. 2015; Rupps et al.
2016; Zhai et al. 2016).
Therefore, identification and characterization of these
genes that participate in the regulatory mechanism of
somatic embryogenesis have opened new windows for
plant biotechnologists. Several genes are specifically acti-
vated or differentially expressed during somatic embryo-
genesis (Chugh and Khurana 2002; Rojas-Herrera et al.
2002). These include somatic embryogenesis receptor
kinase (SERK) (Schmidt et al. 1997; Hu et al. 2005),
LEAFY COTYLEDON (LEC) (Curaba et al. 2004; Gaj et al.
2005; Rupps et al. 2016), BABY BOOM (BBM) (Boutilier
Acta Physiol Plant (2017) 39:194 Page 7 of 17 194
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et al. 2002; Florez et al. 2015), and WUSCHEL (WUS)
(Zuo et al. 2002). Examples of several genes expressed
during somatic embryogenesis in different plant species are
listed in Table 2. Schmidt et al. (1997) found the SERK
expression in an embryogenic culture of Daucus carota
derived from hypocotyl explants for the first time. In
Arabidopsis thaliana, SERK gene expressed and improved
the embryogenic capacity of cultured cells (Feher et al.
2003; Verdeil et al. 2007). Few reports also suggest that,
during somatic embryogenesis induction, SERK gene
encodes a protein, part of the receptor-like kinase-LRR
family, which plays a critical role in signal transduction
(Schmidt et al. 1997; Hecht et al. 2001).
A significant role of SERK gene during somatic
embryogenesis has been observed in both monocotyle-
donous plants such as Dactylis glomerata (Somleva et al.
2000), Ocotea catharinensis (Santa-Catarina et al. 2004),
Oryza sativa (Hu et al. 2005), Triticum aestivum (Singla
et al. 2007), Cocos nucifera (Perez et al. 2015b), Musa
acuminate (Huang et al. 2010), and Zea mays (Zhang et al.
2011), and dicotyledonous plants such as D. carota (Sch-
midt et al. 1997), Arabidopsis thaliana (Hecht et al. 2001;
Salaj et al. 2008), Medicago truncatula (Nolan et al. 2003),
Theobroma cacao (Santos et al. 2005), Citrus unshiu
(Shimada et al. 2005), Vitis vinifera (Schellenbaum et al.
2008), Solanum tuberosum (Sharma et al. 2008), Cyclamen
Table 2 Identified genes expressed during somatic embryogenesis in different plant species
Genes Full name Plant species References
ARF Auxin response factor Dimocarpus longan
Raphanus sativus L.
Zhai et al. (2016), Lin et al. (2015)
AGL15 Agamous-like 15 Brassica napus
Arabidopsis thaliana
Glycine max
Heck et al. (1995), Zheng et al. (2013)
AGO 1 Argonaute 1 Araucaria angustifolia Schlogl et al. (2012a, b)
AGP1 Arabinogalactan protein 1 Lycopersicon
esculentum
Gossypium hirsutum
Pogson and Davies (1995), Poon et al. (2012)
BBM1 Baby Boom 1 Arabidopsis thaliana
Larix decidua
Theobroma cacao
Kulinska-Lukaszek et al. (2012), Rupps et al. (2016),
Florez et al. (2015)
CUC 1 Cup-shaped cotyledon1 Araucaria angustifolia Schlogl et al. (2012a, b)
EMB 1 Daucus carota Wurtele et al. (1993)
GST Glutathione-S-transferase Triticum aestivum Singla et al. (2007)
GLP Germin-like protein Pinus caribaea Morelet Neutelings et al. (1998)
LecKIN S-locus lectin protein kinase Araucaria angustifolia Schlogl et al. (2012a, b)
LEC Leafy cotyledon Larix decidua
Arabidopsis thaliana
Rupps et al. (2016), Gaj et al. (2005), Harada (2001)
PKL Pickle Arabidopsis thaliana
Quercus suber
Ogas et al. (1997); Perez et al. 2015a
PRC1 Protein regulator of cytokinesis Arabidopsis thaliana Chen et al. (2010)
RGP1 Retrograde Golgi transport protein 1 Picea glauca Lippert et al. (2005)
SCR Scarecrow-like Araucaria angustifolia Schlogl et al. (2012a, b)
SERF1 Ethylene response factor Medicago truncatula Mantiri et al. (2008)
SERK Somatic embryogenesis receptor kinase Daucus carota
Oryza sativa
Schmidt et al. (1997), Hu et al. (2005)
VAL1 VP1/ABSCISIC ACID INSENSITIVE 3-LIKE
1
Quercus suber Perez et al. 2015a
WUS Wuschel Arabidopsis thaliana
Coffea canephora
Zuo et al. (2002), Arroyo-Herrera et al. (2008)
WOX4 Wuschel-Related HOMEOBOX4 Coffea canephora
Larix decidua
Nic-Can et al. (2013), Rupps et al. (2016)
194 Page 8 of 17 Acta Physiol Plant (2017) 39:194
123
persicum (Savona et al. 2012), and Trifolium nigrescens
(Pilarska et al. 2016).
LEC genes play a crucial role in maintaining and con-
trolling many aspects of plant embryogenesis and it
encodes transcriptional factors. Two classes of LEC genes
are LEC1, LEC2, and FUSCA3 (FUS3) (Meinke et al.
1994; Luerssen et al. 1998; Harada 2001; Gaj et al. 2005;
Mahdavi-Darvari et al. 2015). Both LEC genes encoded for
regulatory proteins which act primarily in somatic
embryogenesis. (Lotan et al. 1998; Luerssen et al. 1998;
Kwong et al. 2003; Stone et al. 2008) and are crucial to
induce embryo development when expressed ectopically
(Gaj et al. 2005). During plant embryogenesis, these LEC
genes are important for controlling maturation as well as
repressing embryo germination (Parcy et al. 1997; Lotan
et al. 1998; Stone et al. 2008; Yang and Zhang 2010).
WUS is a homeobox gene (encodes a transcription fac-
tor) that maintains the pool of stem cells in the shoot apical
meristem (SAM) (Endrizzi et al. 1996; Laux et al. 1996;
Mayer et al. 1998; Gallois et al. 2004; Bhalla and Singh
2006), and they also encourage the development of somatic
embryos when expressed (Zuo et al. 2002; Gallois et al.
2004). WUS gene is regulated by a feedback loop involving
CLAVATA (CLV) genes, in which CLV gene expresses and
controls the size of the stem cell and seems to be crucial for
maintaining a constant pool of stem cells by repressing
WUS at the transcriptional level (Brand et al. 2000; Schoof
et al. 2000; Waites and Simon 2000; Chen et al. 2009). In
Arabidopsis, ectopic expression of AtWUS stimulates
somatic embryogenesis which has been documented (Zuo
et al. 2002). In Gossypium hirsutum, it has been demon-
strated that during somatic embryogenesis, AtWUS enhance
the conversion of non-embryogenic to embryogenic cells.
(Zheng et al. 2014). WUS is also responsible for activating
LEC genes in Arabidopsis (Wang et al. 2009), and
GhLEC1, GhLEC2, and GhFUS3 genes in G. hirsutum
(Zheng et al. 2014), for somatic embryogenesis induction
and to promote cell differentiation. Furthermore, high
expression of the WUS gene suggests that it is valuable for
the initiation of embryogenesis as a useful gene marker.
BABY BOOM (BBM) is specially expressed in devel-
oping somatic embryos. Ectopically expressed BBM
induces the initiation of somatic embryos or to promote
embryo development (Boutilier et al. 2002). BBM encodes
an AP2/ERF family (APETALA2/ethylene-responsive
factor) domain transcription factors involved in somatic
embryogenesis (Boutilier et al. 2002). In Glycine max (El
Ouakfaoui et al. 2010), as well as A. thaliana and Brassica
napus (Boutilier et al. 2002), BBM overexpressed and
initiated embryogenic callus and establishment of somatic
embryos without the addition of any PGR. Similarly, in
Coffea arabica L., BBM-like gene (CaBBM) used as a
molecular marker during the in vitro embryogenic process
(Silva et al. 2015). Arabidopsis EMBRYOMAKER (EMK)
gene, which encodes an AP2 subfamily, shows a critical
role in the development of embryogenic cells. Ectopic
overexpression of this EMK gene is capable of inducing the
embryo from cotyledons and formation of trichomes on
dedifferentiated tissues (Tsuwamoto et al. 2010). In addi-
tion, microarray-based expression studies also help to
identify BBM target genes.
Germins and germin-like proteins (GLPs) are important
members of a superfamily of functionally diverse proteins
(Dunwell 1998), but structurally is linked to the cupin
superfamily members (Dunwell et al. 2001). GLPs first
observed as a candidate protein marker gene for embryo
germination in wheat embryos during wheat rehydration
(Thompson and Lane 1980). GLPs are known to act as
enzymes, receptors, or structural proteins during somatic
embryogenesis (Domon et al. 1994; Dunwell et al. 2000).
In conifers, GLPs are expressed and responsible for early
embryo development during somatic embryogenesis
(Mathieu et al. 2006). This study shows a potential role for
new GLP gene, LmGER1 in this physiological process.
The expression of LmGER1 has been observed during
somatic embryo maturation. The implications of GLPs in
pine embryogenesis are also reported (Neutelings et al.
1998).
However, available evidence from the little research
suggests that germin-like protein transcripts are reliable
gene markers for the initiation of embryogenesis. A major
upcoming task will be to integrate GLPs role in the initi-
ation of embryogenesis in plants and accelerate their
potential in different biotechnological approaches.
Epigenetic variation during somatic embryogenesis
Epigenetic variation causes phenotypic and genotypic
diversity in plants. In recent years, several reports have
established that epigenetic variation can be influenced by
the in vitro environments at various stages. Although, in
many cases, genetic variation has been observed, vari-
ability in DNA methylation appears to be most common.
In vitro production of plants via somatic embryogenesis
can induce epigenetic variation. The detection of epige-
netic variation during somatic embryogenesis appears to be
mainly focused on DNA methylation as it seems to be one
the best candidate markers to defined mechanism. Several
approaches or techniques for analysis of DNA methylation
are listed in Table 1.
Epigenetic variation in in vitro regenerants propagated
via somatic embryogenesis has been reported in several
plant species such as Corylus avellana L. (Diaz-Sala et al.
1995), Citrus paradisi (Hao et al. 2004), Elaeis guineensis
Jacq. (Jaligot et al. 2004), Rosa hybrida L. (Xu et al. 2004),
Solanum tuberosum L. (Sharma et al. 2007), Vitis vinifera
Acta Physiol Plant (2017) 39:194 Page 9 of 17 194
123
L. (Schellenbaum et al. 2008), Coffea arabica L.
(Menendez-Yuffa et al. 2010; Landey et al. 2015), olive
(Leva et al. 2012), tamarillo (Currais et al. 2013), Solanum
melongena L. cv. Nirrala (Naseer and Mahmood 2014),
Triticale (Machczynska et al. 2015), and Theobroma cacao
L. (Adu-Gyamfi et al. 2016). Epigenetic variation of oil
palm ‘‘mantled’’ is one of the most important examples of
somatic embryo-induced variation (Corley et al. 1986).
This epigenetic variation affects with a decrease in global
DNA methylation associated with the development of
abnormal flowers up to 5% in both male and female
(Corley et al. 1986; Jaligot et al. 2004). In maize, 21 pro-
geny lines derived from tissue cultures of two embryo
sources were examined for DNA methylation changes and
a high level of frequency evidence of demethylation vari-
ation among regenerates was found (Kaeppler and Phillips
1993).
However, epigenetic variation at DNA level has also
been observed in several plant species regenerated via
somatic embryogenesis. For example, in Freesia hybrida,
MSAP analysis shows that DNA methylation alteration in
both CG and CNG levels was almost similar for the direct
(1.1%) and indirect (1.3%) embryogenesis pathways (Gao
et al. 2010). Similarly, in Pinus pinaster, the relative per-
centages of 5mC (5-methylcytosine) in somatic embryos
(23–24% 5mC), and embryo derived plants (17% 5mC),
were very similar as quantified by HPCE (Marum 2009).
Lo Schiavo et al. (1989) recommended that PGRs can
also affect the DNA methylation level in embryonic carrot
cell cultures. However, how the activity of these PGRs
interferes with DNA methylation remains unclear. Fur-
thermore, some antibiotics such as hygromycin, kanamy-
cin, and cefotaxime are known to cause DNA
hypermethylation (Schmitt et al. 1997).
In addition, level of the DNA methylation can be altered
by the cryopreserved embryogenic cell/tissues. It has been
reported that an increased DNA methylation level was
detected in plant obtained from cryopreserved somatic
embryos of Bactris gasipaes when compared to non-cry-
opreserved somatic embryos (Heringer et al. 2013). In T.
cacao L, high levels of phenotypic variability observed in
cryostored somatic embryos may be symptomatic of epi-
genetic change (Adu-Gyamfi et al. 2016).
Epigenetics: biotechnological application
Epigenetics is one of the most promising fields in
biotechnology. In recent years, epigenetic regulation has
been one of the most promising approaches in the current
plant biology because of its potential for food and
biotechnological applications. One of the important
biotechnological approaches for plant productivity and
crop breeding programs has been the utilization of in vitro-
raised plants and their epigenetic effects. Current status of
knowledge on the epigenetic regulation shows that the
influence of these mechanisms plays a key role in plant life
including crop biofortification and plant immunity (Al-
varez et al. 2010; Alvarez-Venegas and De-la-Pena 2016).
Recently, Barraza et al. (2015) found that PvTRX1h gene
responsible for the regulation of plant hormone biosyn-
thesis in the embryogenic calli of common bean and
PvTRX1h gene is down-regulated and capable of differ-
entiate into somatic embryos. In addition, an increased
transcript abundance of a gene coding for a second histone
lysine methyltransferase, PvASHH2h, showed during
down-regulation of PvTRX1h and call attention to that
histone methylation (epigenetic changes) has a potential
role in the biosynthesis of plant hormones during somatic
embryo generation. The authors concluded that this
approach will fill the gaps in plant hormone signalling and
gene regulation of embryogenesis in plants. A detailed
review by De-la-Pena et al. (2015) presents a novel insight
on the key role of chromatin modification in somatic
embryogenesis and how epigenetic regulation mechanisms
could help to improve crop breeding practices and increase
plant productivity.
Ding and Wang (2015) draw attention on the current
understanding of the plant immunity against pathogens and
the role of histone modifications and chromatin remod-
elling mechanisms on it. Plant defense can be affected by
chromatin modifications and remodelling factors that reg-
ulate jasmonic acid and salicylic acid pathway. Few
researches have revealed that how chromatin modifications
and remodelling involved in plant defense (Alvarez et al.
2010; Berr et al. 2012). On the other hand, Kumar et al.
(2015b) reviewed the role of epigenetic silencing in
transgenic research in plant systems used in crop
improvement. In epigenetic silencing, expression of genes
is regulated through modification DNA, RNA, and histone
proteins. It acts as an expression modulator and helps for
defending host genomes against the effects of viral infec-
tion and transposable elements. Epigenetic silencing in
transgenic plants was first discovered in transgenic tobacco
due to the interaction between two homologous promoters
(Matzke et al. 1989). Paul et al. (2015) also discussed
recent discoveries on nutrient homeostasis regulation in
plants by miRNA. In the review, the authors highlighted
the role of several miRNAs in nutrient deficiencies in
plants and how nutrient-related miRNA and their gene
regulation technology could be used in crop improvement
strategies and biotechnological research in the future. In
addition, epigenetics research has a crucial role in
improving of nutritional value in crop (Angaji et al. 2010),
increasing stress tolerance in plants (Manavalan et al. 2012;
Garg et al. 2015), improvement of plant disease resistance
in plants (Duan et al. 2012; Catoni et al. 2013), in plant
194 Page 10 of 17 Acta Physiol Plant (2017) 39:194
123
heat responses (Liu et al. 2015), in plant–microbe inter-
actions (Zhu et al. 2016), nutrient deficiency responses in
plants (Sirohi et al. 2016), and manipulating plant archi-
tecture, flower colour, flowering time, fruit development,
and in forestry for wood quality (Guan et al. 2016).
Therefore, epigenetic mechanisms will be an essential and
inevitable in the near future, and can be consider as an
innovative approach for biotechnological applications in
the 21st century.
Conclusion and future perspectives
Somatic embryogenesis is an important developmental
pathway in plants and has considerable interest for
biotechnological application, in which competent somatic
cells can dedifferentiate to a totipotent embryonic cell
under appropriate conditions, build up into somatic
embryos and passing through different developmental
stages, and, finally, give rise to complete plantlet formation
(Arnold et al. 2002). During this complex developmental
pathway of embryogenesis, cells have to dedifferentiate
and reprogramme their patterns of gene expression at dif-
ferent levels (Yang and Zhang 2010).
During somatic embryogenesis in plants, regulation of
epigenetic mechanisms (methylation of DNA, chromatin
remodelling, and microRNAs) regulate gene expression.
To differentiate between both non-embryogenic and
embryogenic cells, these mechanisms of epigenetic regu-
lation could be utilized as a candidate marker. Methylation
of DNA is a significant epigenetic mechanism for the
epigenetic regulation that has been studied with various
methods. Although it is essential to discover the key role of
the different methyltransferases (MET) in somatic
embryogenesis, because it is still unclear among different
enzymes which one is involved in plant embryogenesis. In
future, regulated DNA methylation will serve as an
essential biotechnological tool to enhance quantity and
improve quality of plants. Especially, it will be very useful
in technology of plant biofactories for mass production of
proteins, vaccines, and others biologically active peptides.
Future works are necessary to explain how DNA methy-
lation is maintained and established, and how gene-body
methylation is involved in gene transcription regulation in
plant embryogenesis.
We note that there is evidence that chromatin remod-
elling could be utilized as a potential mechanisms for
identification of cellular dedifferentiation, genome stability
maintenance, and plant development. It was found that
BBM1 is vital for morphogenesis and cell proliferation
during embryogenesis (Boutilier et al. 2002), while LEC1
and WOX4 are crucial in the initial phase of cell differ-
entiation (Lotan et al. 1998; Ji et al. 2010; Suer et al. 2011).
In future, research on how chromatin remodelling is
involved in somatic embryogenesis will open new
approaches and significantly contribute to our understand-
ing of the molecular mechanisms, which may help as an
exciting interaction model and finally be applicable to
enhance plant productivity.
miRNAs also play a crucial roles as important regulators
in epigenetic processes and also have a profound effect in
controlling gene expression in plants. Like miR156 acti-
vated throughout early somatic embryogenesis and also
shows a critical role in the initial phase of cell differenti-
ation. Therefore, for miRNAs and miRNAs-mediated gene
silencing mechanism, identification and functional char-
acterization during somatic embryogenesis are expected to
offer innovative understandings in totipotency of plant cell
and to explore for different approaches to sustain and
improve the capacity of embryogenic cells related to plant
embryo development. However, more innovative study is
required to identify and control the regulation of epigenetic
mechanism during somatic embryogenesis-related miR-
NAs and its significance in the success of plant develop-
ment. Future work should focus on how epigenetic
mechanisms in somatic embryogenesis could help to
increase breeding practices and improve plant productivity.
Author contribution statement VK conceived the idea,
collected the data, and wrote the manuscript. JVS edited
and corrected the manuscript. All the authors read and
approved the final version of the manuscript.
Acknowledgements VK is grateful to the Claude Leon Foundation
and the University of KwaZulu-Natal, South Africa for the financial
support in the form of postdoctoral fellowship. We also thank the
anonymous reviewers for their suggestions which help to improve the
manuscript.
Compliance with ethical standards
Conflict of interest Authors have no competing interests in the
manuscript.
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