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

rcpgd@ukzn.ac.za

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

123

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

123

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)

194 Page 4 of 17 Acta Physiol Plant (2017) 39:194

123

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

123

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

123

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

123

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.

References

Adu-Gyamfi R, Wetten A, Rodrıguez L, Carlos M (2016) Effect of

cryopreservation and post-cryopreservation somatic embryoge-

nesis on the epigenetic fidelity of cocoa (Theobroma cacao L.).

PLoS One 11:e0158857

Alvarez ME, Nota F, Cambiagno DA (2010) Epigenetic control of

plant immunity. Mol Plant 11:563–576

Alvarez-Venegas R, De-la-Pena C (2016) Editorial: recent advances

of epigenetics in crop biotechnology. Front Plant Sci 7:413

Angaji SA, Hedayati S, Poor RH, Madani S (2010) Application of

RNA interference in plants. Plant Omics 3:77–84

Arnold SV, Sabala I, Bozhkov P, Dyachok J, Filonova L (2002)

Developmental pathways of somatic embryogenesis. Plant Cell

Tissue Organ Cult 69:233–249

Arroyo-Herrera A, Gonzalez AK, Moo RC, Quiroz-Figueroa FR,

Loyola-Vargas VM, Rodriguez-Zapata LC, Burgeff D’Hondt C,

Acta Physiol Plant (2017) 39:194 Page 11 of 17 194

123

Suarez-Solıs VM, Castano E (2008) Expression of WUSCHEL

in Coffea canephora causes ectopic morphogenesis and increases

somatic embryogenesis. Plant Cell Tissue Organ Cult

94:171–180

Avivi Y, Morad V, Ben-Meir H, Zhao J, Kashkush K, Tzfira T,

Citovsky V, Grafi G (2004) Reorganization of specific chromo-

somal domains and activation of silent genes in plant cells

acquiring pluripotentiality. Dev Dyn 230:12–22

Barraza A, Cabrera-Ponce JL, Gamboa-Becerra R, Luna-Martinez F,

Winkler R, Alvarez-Venegas R (2015) The Phaseolus vulgaris

PvTRX1h gene regulates plant hormone biosynthesis in embryo-

genic callus from common bean. Front Plant Sci 6:577

Bartel DP (2004) MicroRNAs: genomics, biogenesis, mechanism, and

function. Cell 116:281–297

Bednarek P, Orlowska R, Koebner R, Zimny J (2007) Quantification

of the tissue-culture induced variation in barley (Hordeum

vulgare L.). BMC Plant Biol 7:10

Berger SL, Kouzarides T, Shiekhattar R, Shilatifard A (2009) An

operational definition of epigenetics. Genes Dev 23:781–783

Berr A, Menard R, Heitz T, Shen WH (2012) Chromatin modification

and remodelling: a regulatory landscape for the control of

Arabidopsis defence responses upon pathogen attack. Cell

Microbiol 14:829–839

Bhalla PL, Singh MB (2006) Molecular control of stem cell

maintenance in shoot apical meristem. Plant Cell Rep

25:249–256

Boutilier K, Offringa R, Sharma VK, Kieft H, Ouellet T, Zhang L,

Hattori J, Liu CM, van Lammeren AAM, Miki BLA, Custers

JBM, Campagne MML (2002) Ectopic expression of BABY

BOOM triggers a conversion from vegetative to embryonic

growth. Plant Cell 14:1737–1749

Bouyer D, Roudier F, Heese M, Andersen E, Gey D, Nowack MK,

Goodrich J, Renou JP, Grini PE, Colot V, Schnittger A (2011)

Polycomb repressive complex 2 controls the embryo-to-seedling

phase transition. PLoS Genet 7:e1002243

Brand U, Fletcher JC, Hobe M, Meyerowitz EM, Simon R (2000)

Dependence of stem cell fate in Arabidopsis on a feedback loop

regulated by CLV3 activity. Science 289:617–619

Braybrook SA, Stone SL, Park S, Bui AQ, Le BH, Fischer RL,

Goldberg RB, Harada JJ (2006) Genes directly regulated by

LEAFY COTYLEDON2 provide insight into the control of

embryo maturation and somatic embryogenesis. Proc Natl Acad

Sci USA 103:3468–3473

Brown PTH, Kyozuka J, Sukekiyo Y, Kimura Y, Shimamoto K, Lorz

H (1990) Molecular changes in protoplast-derived rice plants.

Mol Gen Genet 223:324–328

Butler PJ (1983) The folding of chromatin. CRC Crit Rev Biochem

15:57–91

Cao X, Jacobsen SE (2002) Locus-specific control of asymmetric and

CpNpG methylation by the DRM and CMT3 methyltransferase

genes. Proc Natl Acad Sci USA 99:16491–16498

Catoni M, Lucioli A, Doblas-Ibanez P, Accotto GP, Vaira AM (2013)

From immunity to susceptibility: virus resistance induced in

tomato by a silenced transgene is lost as TGS overcomes PTGS.

Plant J 75:941–953

Chakrabarty D, Yu KW, Paek KY (2003) Detection of DNA

methylation changes during somatic embryogenesis of Siberian

ginseng (Eleutherococcus senticosus). Plant Sci 165:61–68

Chavez-Hernandez EC, Alejandri-Ramırez ND, Juarez-Gonzalez VT,

Dinkova TD (2015) Maize miRNA and target regulation in

response to hormone depletion and light exposure during somatic

embryogenesis. Front Plant Sci 6:555

Chen SK, Kurdyukov S, Kereszt A, Wang XD, Gressho PM, Rose RJ

(2009) The association of homeobox gene expression with stem

cell formation and morphogenesis in cultured Medicago trun-

catula. Planta 230:827–840

Chen D, Molitor A, Liu C, Shen WH (2010) The Arabidopsis PRC1-

like ring-finger proteins are necessary for repression of embry-

onic traits during vegetative growth. Cell Res 20:1332–1344

Chen CJ, Liu Q, Zhang YC, Qu LH, Chen YQ, Gautheret D (2011)

Genome-wide discovery and analysis of microRNAs and other

small RNAs from rice embryogenic callus. RNA Biol 8:538–547

Chugh A, Khurana P (2002) Gene expression during somatic

embryogenesis—recent advances. Curr Sci 86:715–730

Corley RHV, Lee CH, Law LH, Wong CY (1986) Abnormal flower

development in oil palm clones. Planter (Kuala Lumpur)

62:233–240

Cristofolini C, do Nascimento Vieira L, de Freitas Fraga H, da Costa

I, Guerra M, Pescador R (2014) DNA methylation patterns and

karyotype analysis of off-type and normal phenotype somatic

embryos of feijoa. Theor Exp Plant Physiol 26:217–224

Curaba J, Moritz T, Blervaque R, Parcy F, Raz V, Herzog M, Vachon

G (2004) AtGA3ox2, a key gene responsible for bioactive

gibberellin biosynthesis, is regulated during embryogenesis by

LEAFY COTYLEDONS2 and FUS3 in Arabidopsis. Plant

Physiol 136:3660–3669

Currais L, Loureiro J, Santos C, Canhoto JM (2013) Ploidy stability in

embryogenic cultures and regenerated plantlets of tamarillo.

Plant Cell Tissue Organ Cult 114:149–159

De-la-Pena C, Nic-Can GI, Galaz-Avalos RM, Avilez-Montalvo R,

Loyola-Vargas VM (2015) The role of chromatin modifications

in somatic embryogenesis in plants. Front Plant Sci 6:635

Diaz-Sala C, Rey M, Boronat A, Besford R, Rodriguez R (1995)

Variations in the DNA methylation and polypeptide patterns of

adult hazel (Corylus avellana L.) associated with sequential

in vitro subcultures. Plant Cell Rep 15:218–221

Ding B, Wang GL (2015) Chromatin versus pathogens: the function

of epigenetics in plant immunity. Front Plant Sci 6:675

Domon JM, Meyer Y, Faye L, David A, David H (1994) Extracellular

(g1yco) proteins in embryogenic and non-embryogenic cell lines

of Caribbean pine. Comparison between phenotypes of stage one

somatic embryos. Plant Physiol Biochem 32:1–11

Duan CG, Wang CH, Guo HS (2012) Application of RNA silencing

to plant disease resistance. Silence 3:5

Dunwell JM (1998) Cupins: a new superfamily of functionally diverse

proteins that include germins and plant storage proteins.

Biotechnol Genet Eng Rev 15:1–32

Dunwell J, Khuri S, Gane P (2000) Microbial relatives of the seed

storage proteins of higher plants: conservation of structure and

diversification of function during evolution of the cupin super-

family. Microbiol Mol Biol Rev 64:153–179

Dunwell JM, Culham A, Carter CE, Sosa-Aguirre CR, Goodenough

PW (2001) Evolution of functional diversity in the cupin

superfamily. Trends Biochem Sci 26:740–746

El Ouakfaoui S, Schnell J, Abdeen A, Colville A, Labbe H, Han S,

Baum B, Laberge S, Miki B (2010) Control of somatic

embryogenesis and embryo development by AP2 transcription

factors. Plant Mol Biol 74:313–326

Elhiti M, Stasolla C, Wang A (2013) Molecular regulation of plant

somatic embryogenesis. In Vitro Cell Dev Biol Plant

49:631–642

Elmeer KES (2013) Factors regulating somatic embryogenesis in

plants. In: Junaid A, Srivastava PS, Sharma MP (eds) Somatic

embryogenesis and gene expression. Narosa Publishing House,

New Delhi, pp 56–81

Endrizzi K, Moussian B, Haecker A, Levin JZ, Laux T (1996) The

shoot meristemless gene is required for maintenance of undif-

ferentiated cells in Arabidopsis shoot and floral meristems and

acts at a different regulatory level than the meristem genes

WUSCHEL and ZWILLE. Plant J 10:967–979

Exner V, Hennig L (2008) Chromatin rearrangements in develop-

ment. Curr Opin Plant Biol 11:64–69

194 Page 12 of 17 Acta Physiol Plant (2017) 39:194

123

Feher A (2015) Somatic embryogenesis—stress-induced remodeling

of plant cell fate. Biochim Biophys Acta 1849:385–402

Feher A, Pasternak T, Dudits D (2003) Transition of somatic plant

cells to an embryogenic state. Plant Cell Tissue Organ Cult

74:201–228

Fiuk A, Bednarek PT, Rybczynski JJ (2010) Flow cytometry, HPLC-

RP, and metAFLP analyses to assess genetic variability in

somatic embryo-derived plantlets of Gentiana pannonica Scop.

Plant Mol Biol Rep 28:413–420

Florez SL, Erwin RL, Maximova SN, Guiltinan MJ, Curtis WR

(2015) Enhanced somatic embryogenesis in Theobroma cacao

using the homologous BABY BOOM transcription factor. BMC

Plant Biol 15:121

Fraga HPF, Vieira LN, Caprestano CA, Steinmacher DA, Micke GA,

Spudeit DA, Rosete Pescador R, Guerra MP (2012) 5-Azacy-

tidine combined with 2,4-D improves somatic embryogenesis of

Acca sellowiana (O. Berg) Burret by means of changes in global

DNA methylation levels. Plant Cell Rep 31:2165–2176

Gaj MD, Zhang S, Harada JJ, Lemaux P (2005) Leafy cotyledon

genes are essential for induction of somatic embryogenesis of

Arabidopsis. Planta 222:977–988

Gallois JL, Nora FR, Mizukami Y, Sablowski R (2004) WUSCHEL

induces shoot stem cell activity and developmental plasticity in

the root meristem. Genes Dev 18:375–380

Gao X, Yang D, Cao D, Ao M, Sui X, Wang Q, Kimatu J, Wang L

(2010) In vitro micropropagation of Freesia hybrida and the

assessment of genetic and epigenetic stability in regenerated

plantlets. J Plant Growth Regul 29:257–267

Garg R, Chevala VVSN, Shankar R, Jain M (2015) Divergent DNA

methylation patterns associated with gene expression in rice

cultivars with contrasting drought and salinity stress response.

Sci Rep 5:14922

Gazzarrini S, Tsuchiya Y, Lumba S, Okamoto M, McCourt P (2004)

The transcription factor FUSCA3 controls developmental timing

in Arabidopsis through the hormones gibberellin and abscisic

acid. Dev Cell 7:373–385

Gillis K, Gielis J, Peeters H, Dhooghe E, Oprins J (2007) Somatic

embryogenesis from mature Bambusa balcooa Roxburgh as

basis for mass production of elite forestry bamboos. Plant Cell

Tissue Organ Cult 91:115–123

Grafi G, Ben-Meir H, Avivi Y, Moshe M, Dahan Y, Zemach A (2007)

Histone methylation controls telomerase-independent telomere

lengthening in cells undergoing dedifferentiation. Dev Biol

306:838–846

Guan Y, Li SG, Fan XF, Su ZH (2016) Application of somatic

embryogenesis in woody plants. Front Plant Sci 7:938

Haig D (2004) The (dual) origin of epigenetics. Cold Spring Harb

Symp Quant Biol 69:67–70

Han SK, Wu MF, Cui S, Wagner D (2015) Roles and activities of

chromatin remodeling ATPases in plants. Plant J 83:62–77

Hanani LR, Floh EIS, Fungaro MHP, Santa-Catarina C, Matias de

Paula F, Viana AM, Vieira MLC (2010) Methylation patterns

revealed by MSAP profiling in genetically stable somatic

embryogenic cultures of Ocotea catharinensis (Lauraceae).

In Vitro Cell Dev Biol Plant 46:368–377

Hao YJ, Wen XP, Deng XX (2004) Genetic and epigenetic

evaluations of citrus calluses recovered from slow-growth

culture. J Plant Physiol 161:479–484

Harada JJ (2001) Role of Arabidopsis LEAFY COTYLEDON genes

in seed development. J Plant Physiol 158:405–409

He X, Chen T, Zhu J (2011) Regulation and function of DNA

methylation in plants and animals. Cell Res 21:442–465

Hecht V, Vielle-Calzada JP, Hartog MV, Schmidt EDL, Boutilier K,

Grossniklaus U, De Vries SC (2001) The Arabidopsis somatic

embryogenesis receptor kinase 1 gene is expressed in developing

ovules and embryos and enhances embryogenic competence in

culture. Plant Physiol 127:803–816

Heck GR, Perry SE, Nichols KW, Fernandez DE (1995) AGL15, a

MADS domain protein expressed in developing. Plant Cell

7:1271–1282

Heringer AS, Steinmacher DA, Fraga HP, Vieira LN, Ree JF, Guerra

MP (2013) Global DNA methylation profiles of somatic embryos

of peach palm (Bactris gasipaes Kunth) are influenced by

cryoprotectants and droplet-vitrification cryopreservation. Plant

Cell Tissue Organ Cult 114:365–372

Holec S, Berger F (2012) Polycomb group complexes mediate

developmental transitions in plants. Plant Physiol 158:35–43

Hu H, Xiong L, Yang Y (2005) Rice SERK1 gene positively regulates

somatic embryogenesis of cultured cells and host defense

response against fungal infection. Planta 222:107–117

Huang X, Lu XY, Zhao JT, Chen JK, Dai XM, Xiao W, Chen YP,Chen YF, Huang XL (2010) MaSERK1 gene expression

associated with somatic embryogenic competence and disease

resistance response in banana (Musa spp.). Plant Mol Biol Rep

28:309–316

Ikeuchi M, Iwase A, Rymen B, Harashima H, Shibata M, Ohnuma M,

Breuer C, Morao AK, de Lucas M, de Veylder L, Goodrich J,

Brady SM, Roudier F, Sugimoto K (2015) PRC2 represses

dedifferentiation of mature somatic cells in Arabidopsis. Nat

Plants 1:15089

Ikeuchi M, Ogawa Y, Iwase A, Sugimoto K (2016) Plant regener-

ation: cellular origins and molecular mechanisms. Development

143:1442–1451

Jaligot E, Beule T, Baurens FC, Billotte N, Rival A (2004) Search for

methylation-sensitive amplification polymorphisms associated

with the ‘mantled’ variant phenotype in oil palm (Elaeis

guineensis Jacq.). Genome 47:224–228

Jarillo JA, Pineiro M, Cubas P, Martınez-Zapater JM (2009)

Chromatin remodeling in plant development. Int J Dev Biol

53:1581–1596

Ji J, Strable J, Shimizu R, Koening D, Sinha N, Scanlon MJ (2010)

WOX4 promotes procambial development. Plant Physiol

152:1346–1356

Jia X, Yan J, Tang G (2011) MicroRNA-mediated DNA methylation

in plants. Front Biol 6:133–139

Jimenez VM (2005) Involvement of plant hormones and plant growth

regulators on in vitro somatic embryogenesis. Plant Growth

Regul 47:91–110

Jimenez VM, Bangerth F (2001) Endogenous hormone concentrations

and embryogenic callus development in wheat. Plant Cell Tissue

Organ Cult 67:37–46

Jones-Rhoades WM, Bartel DP, Bartel B (2006) MicroRNAs and

their regulatory roles in plants. Annu Rev Plant Biol 57:19–53

Joshi R, Joshi PR, Aramod Kumar PK (2013) Regulation of somatic

embryogenesis in crops: a review. Agric Rev 34:1–20

Junker A, Monke G, Rutten T, Keilwagen J, Seifert M, Thi TMN,

Renou JP, Balzergue S, Viehover P, Hahnel U, Ludwig-Muller J,

Altschmied L, Conrad U, Weisshaar B, Baumlein H (2012)

Elongation-related functions of LEAFY COTYLEDON1 during

the development of Arabidopsis thaliana. Plant J 71:427–442

Kaeppler SM, Phillips RL (1993) Tissue culture-induced DNA

methylation variation in maize. Proc Natl Acad Sci USA

90:8773–8776

Klimaszewska K, Noceda C, Pelletier G, Label P, Rodriguez R, Lelu-

Walter MA (2009) Biological characterization of young and

aged embryogenic cultures of Pinus pinaster (Ait.). In Vitro Cell

Dev Biol Plant 45:20–33

Kulinska-Lukaszek K, Tobojka M, Adamiok A, Kurczynska EU

(2012) Expression of the BBM gene during somatic embryoge-

nesis of Arabidopsis thaliana. Biol Plant 56:389–394

Acta Physiol Plant (2017) 39:194 Page 13 of 17 194

123

Kumar V, Moyo M, Van Staden J (2015a) Somatic embryogenesis of

Pelargonium sidoides DC. Plant Cell Tissue Organ Cult

121:571–577

Kumar R, Anunanthini P, Sathishkumar R (2015b) Epigenetic

silencing in transgenic plants. Front Plant Sci 6:693

Kumar V, Moyo M, Van Staden J (2016) Enhancing plant regener-

ation of Lachenalia viridiflora, a critically endangered orna-

mental geophyte with high floricultural potential. Sci Hortic

211:263–268

Kumar V, Moyo M, Van Staden J (2017) Somatic embryogenesis in

Hypoxis hemerocallidea: an important African medicinal plant.

South Afr J Bot 108:331–336

Kwong RM, Bui AQ, Lee H, Kwong LW, Fischer RL, Goldberg RB,

Harada JJ (2003) LEAFY COTYLEDON1-LIKE defines a class

of regulators essential for embryo development. Plant Cell

15:5–18

Lafos M, Kroll P, Hohenstatt ML, Thorpe FL, Clarenz O, Schubert D

(2011) Dynamic regulation of H3K27 trimethylation during

Arabidopsis differentiation. PLoS Genet 7:e1002040

Landey RB, Cenci A, Guyot R, Bertrand B, Georget F, Dechamp E,

Herrera JC, Aribi J, Lashermes P, Etienne H (2015) Assessment

of genetic and epigenetic changes during cell culture ageing and

relations with somaclonal variation in Coffea arabica. Plant Cell

Tissue Organ Cult 122:517–531

Laux T, Mayer KFX, Berger J, Jurgens G (1996) The WUSCHEL

gene is required for shoot and floral meristem integrity in

Arabidopsis. Development 122:87–96

Lee Y, Jeon K, Lee JT, Kim S, Kim VN (2002) MicroRNA

maturation: stepwise processing and subcellular localization.

EMBO J 21:4663–4670

Leljak-Levanic D, Bauer N, Mihaljevic S, Jelaska S (2004) Changes

in DNA methylation during somatic embryogenesis in Cucurbita

pepo L. Plant Cell Rep 23:120–127

Leva AR, Petruccelli R, Rinaldi LMR (2012) Somaclonal variation in

tissue culture: a case study with olive. In: Leva AR, Rinaldi

LMR (eds) Recent advances in plant in vitro culture. INTECH

Open Access Publisher, Croatia, pp 123–150

Levanic DL, Mihaljevic S, Jelaska S (2009) Variations in DNA

methylation in Picea omorika (Panc) Purk. embryogenic tissue

and the ability for embryo maturation. Prop Orn Plants 9:3–9

Li X, Yu X, Wang N, Feng Q, Dong Z, Liu L, Shen J, Liu B (2007)

Genetic and epigenetic instabilities induced by tissue culture in

wild barley (Hordeum brevisubulatum (Trin.) Link). Plant Cell

Tissue Organ Cult 90:153–168

Li H, Zhao X, Dai H, Wu W, Mao W, Zhang Z (2012a) Tissue culture

responsive microRNAs in strawberry. Plant Mol Biol Rep

30:1047–1054

Li T, Chen J, Qiu S, Zhang Y, Wang P, Yang L, Lu Y, Shi J (2012b)

Deep sequencing and microarray hybridization identify con-

served and species-specific microRNAs during somatic embryo-

genesis in hybrid yellow poplar. PLoS One 7:e43451

Lin Y, Lai Z (2013) Comparative analysis reveals dynamic changes in

miRNAs and their targets and expression during somatic

embryogenesis in longan (Dimocarpus longan Lour.). PLoS

One 8:e60337

Lin Y, Lai Z, Tian Q, Lin L, Lai R, Yang M, Zhang D, Chen Y,

Zhang Z (2015) Endogenous target mimics down-regulate

miR160 mediation of ARF10, -16 and -17 cleavage during

somatic embryogenesis in Dimocarpus longan Lour. Front Plant

Sci 6:956

Lippert D, Zhuang J, Ralph S, Ellis DE, Gilbert M, Olafson R, Ritland K,

Ellis B, Douglas CJ, Bohlmann J (2005) Proteome analysis of early

somatic embryogenesis in Picea glauca. Proteomics 5:461–473

Liu B, Li P, Li X, Liu C, Cao S, Chu C, Cao X (2005) Loss of

function of OsDCL1 affects microRNA accumulation and causes

developmental defects in rice. Plant Physiol 139:296–305

Liu J, Feng L, Li J, He Z (2015) Genetic and epigenetic control of

plant heat responses. Front Plant Sci 6:267

Lo Schiavo F, Pitto L, Giuliano G, Torti G, Nuti Ronchi V, Marazzati

D, Vergara R, Orselli S, Terzi M (1989) DNA methylation of

embryogenic carrot cell cultures and its variations as caused by

mutation, differentiations, hormones and hypomethylating drugs.

Theor Appl Genet 77:325–331

Lopez CMR, Wetten AC, Wilkinson MJ (2010) Progressive erosion

of genetic and epigenetic variation in callus-derived cocoa

(Theobroma cacao) plants. New Phytol 186:856–868

Lotan T, Ohto M, Matsudaira YK, West MAL, Lo R, Kwong RW,

Yamagishi K, Fischer RL, Goldberg RB, Harada JJ (1998)

Arabidopsis LEAFY COTYLEDON1 is sufficient to induce

embryo development in vegetative cells. Cell 93:1195–1205

Loyola-Vargas VM, De-la-Pena C, Galaz-Avalos RM, Quiroz-

Figueroa FR (2008) Plant tissue culture. An intemporal set of

tools. In: Walker JM, Rapley R (eds) Protein and cell biometh-

ods handbook. Humana Press, Totowa, pp 875–904

Luerssen H, Kirik V, Herrman P, Misera S (1998) FUSCA3encodesa

protein with a conserved Vp1/AB13-like B3 domain which is of

functional importance for the regulation of seed maturation in

Arabidopsis thaliana. Plant J 15:755–764

Luo YC, Zhou H, Li Y, Chen JY, Yang JH, Chen YQ, Qu LH (2006)

Rice embryogenic calli express a unique set of microRNAs,

suggesting regulatory roles of microRNAs in plant postembryo-

genic development. FEBS Lett 580:5111–5116

Machczynska J, Zimny J, Bednarek PT (2015) Tissue culture-induced

genetic and epigenetic variation in triticale (9Triticosecale spp.

Wittmack ex A. Camus 1927) regenerants. Plant Mol Biol

89:279–292

Mahdavi-Darvari F, Noor NM, Ismanizan I (2015) Epigenetic

regulation and gene markers as signals of early somatic

embryogenesis. Plant Cell Tissue Organ Cult 120:407–422

Manavalan LP, Chen X, Clarke J, Salmeron J, Nguyen HT (2012)

RNAi-mediated disruption of squalene synthase improves

drought tolerance and yield in rice. J Exp Bot 63:163–175

Mantiri FR, Kurdyukov S, Lohar DP, Sharopova N, Saeed NA, Wang

XD, Vandenbosch KA, Rose RJ (2008) The transcription factor

MtSERF1 of the ERF subfamily identified by transcriptional

profiling is required for somatic embryogenesis induced by auxin

plus cytokinin in Medicago truncatula. Plant Physiol

146:1622–1636

Marum L (2009) Genetic and epigenetic approaches to evaluate

stability during in vitro propagation of Pinus pinaster Ait. PhD

Thesis. Instituto de Tecnologia Quımica e Biologica Universi-

dade Nova de Lisboa

Mathieu M, Lelu-Walter MA, Blervacq AS, David H, Hawkins S,

Neutelings G (2006) Germin-like genes are expressed during

somatic embryogenesis and early development of conifers. Plant

Mol Biol 61:615–627

Matzke MA, Primig M, Trnovsky J, Matzke AJM (1989) Reversible

methylation and inactivation of marker genes in sequentially

transformed tobacco plants. EMBO J 8:643–649

Mayer KF, Schoof H, Haecker A, Lenhard M, Jurgens G, Laux T

(1998) Role of WUSCHEL in regulating stem cell fate in the

Arabidopsis shoot meristem. Cell 95:805–815

Meinke DW, Franzmann LH, Nickle TC, Yeung EC (1994) Leafy

cotyledon mutants of Arabidopsis. Plant Cell 6:1049–1064

Menendez-Yuffa A, Barry-Etienne D, Bertrand B, Georget F, Etienne

H (2010) A comparative analysis of the development and quality

of nursery plants derived from somatic embryogenesis and from

seedlings for large-scale propagation of coffee (Coffea arabica

L.). Plant Cell Tissue Organ Cult 102:297–307

Miguel C, Marum L (2011) An epigenetic view of plant cells cultured

in vitro: somaclonal variation and beyond. J Exp Bot

62:3713–3725

194 Page 14 of 17 Acta Physiol Plant (2017) 39:194

123

Milutinovic S, Zhuang Q, Niveleau A, Szyf M (2003) Epigenomic

stress response Knockdown of DNA methyltransferase 1 triggers

an intra-S-phase arrest of DNA replication and induction of

stress response genes. J Biol Chem 278:14985–14995

Mordhorst AP, Toonen MA, de Vries SC, Meinke D (1997) Plant

embryogenesis. Crit Rev Plant Sci 16:535–576

Munksgaard D, Mattsson O, Okkles FT (1995) Somatic embryo

development in carrot is associated with an increase in levels of

S-adenosylmethionine, S-adenosylhomocysteine and DNA

methylation. Physiol Plant 93:5–10

Naseer S, Mahmood T (2014) Tissue culture and genetic analysis of

somaclonal variations of Solanum melongena L. cv. Nirrala.

Cent Eur J Biol 9:1182–1195

Neelakandan AK, Wang K (2012) Recent progress in the under-

standing of tissue culture-induced genome level changes in

plants and potential applications. Plant Cell Rep 31:597–620

Neutelings G, Domon JM, Membre N, Bernier F, Meyer Y, David A,

David H (1998) Characterization of a germin-like protein gene

expressed in somatic and zygotic embryos of pine (Pinus

caribaea Morelet). Plant Mol Biol 38:1179–1190

Nic-Can GI, De-la-Pena C (2014) Epigenetic advances on somatic

embryogenesis of agronomical and important crops. Epigenetics

in plants of agronomic importance: fundamentals and applica-

tions. Springer, New York, pp 91–109

Nic-Can GI, Lopez-Torres A, Barredo-Pool FA, Wrobel K, Loyola-

Vargas VM, Rojas-Herrera R, De-la-Pena C (2013) New insights

into somatic embryogenesis: LEAFY COTYLEDON1, BABY

BOOM1 and WUSCHEL-RELATED HOMEOBOX4 are epi-

genetically regulated in Coffea canephora. PLoS One 8:e72160

Noceda C, Salaj T, Perez M, Viejo M, Canal J, Salaj J, Rodriguez R

(2009) DNA methylation and decrease on free polyamines is

associated with the embryogenic capacity of Pinus nigra Arn.

Cell Cult Trees 23:1285–1293

Nodine MD, Bartel DP (2010) MicroRNAs prevent precocious gene

expression and enable pattern formation during plant embryo-

genesis. Genes Dev 24:2678–2692

Nolan KE, Irwanto RR, Rose RJ (2003) Auxin up-regulates

MtSERK1 expression in both Medicago truncatula root-forming

and embryogenic cultures. Plant Physiol 133:218–230

Ogas J, Cheng JC, Sung ZR, Somerville C (1997) Cellular differen-

tiation regulated by gibberellin in the Arabidopsis thaliana

pickle mutant. Science 277:91–94

Palmgren G, Mattsson O, Okkels FT (1991) Specific levels of DNA

methylation in various tissues, cell lines, and cell types of

Daucus carota. Plant Physiol 95:174–178

Parcy F, Valon C, Kohara A, Misera S, Giraudat J (1997) The

ABSCISIC ACID-INSENSITIVE3, FUSCA3, and LEAFY

COTYLEDON1 loci act in concert to control multiple aspects

of Arabidopsis seed development. Plant Cell 9:1265–1277

Park W, Li J, Song R, Messing J, Chen X (2002) CARPEL

FACTORY, a Dicer homolog, and HEN1, a novel protein, act in

microRNA metabolism in Arabidopsis thaliana. Curr Biol

12:1484–1495

Paul S, Datta SK, Datta K (2015) miRNA regulation of nutrient

homeostasis in plants. Front Plant Sci 6:232

Perez M, Canal MJ, Toorop PE (2015a) Expression analysis of

epigenetic and abscisic acid-related genes during maturation of

Quercus suber somatic embryos. Plant Cell Tissue Organ Cult

121:353–366

Perez M, Viejo M, LaCuesta M, Toorop P, Canal MJ (2015b)

Epigenetic and hormonal profile during maturation of Quercus

suber L. somatic embryos. J Plant Physiol 173:51–61

Pilarska M, Malec P, Salaj J, Bartnicki F, Konieczny R (2016) High

expression of SOMATIC EMBRYOGENESIS RECEPTOR-LIKEKINASE coincides with initiation of various developmental

pathways in in vitro culture of Trifolium nigrescens. Protoplasma

253:345–355

Pogson BJ, Davies C (1995) Characterization of a cDNA encoding

the protein moiety of a putative arabinogalactan protein from

Lycopersicon esculentum. Plant Mol Biol 28:347–352

Poon S, Heath RL, Clarke AE (2012) A chimeric arabinogalactan

protein promotes somatic embryogenesis in cotton cell cul-

ture1,[W][OA]. Plant Physiol 160:684–695

Qiao M, Xiang F (2013) A set of Arabidopsis thaliana miRNAs

involve shoot regeneration in vitro. Plant Signal Behav 8:e23479

Quiroz-Figueroa FR, Rojas-Herrera R, Galaz-Avalos RM, Loyola

Vargas VM (2006) Embryo production through somatic embryo-

genesis ca be used to study cell differentiation in plants. Plant

Cell Tissue Organ Cult 86:285–301

Reinert J (1959) Uber die Kontrolle der Morphogenese und die

Induktion von Adventivembryonen an Gewebekulturen aus

Karotten. Planta 53:318–333

Reyes JC (2006) Chromatin modifiers that control plant development.

Curr Opin Plant Biol 9:21–27

Rhoades MW, Reinhart BJ, Lim LP, Burge CB, Bartel B, Bartel DP

(2002) Prediction of plant microRNA targets. Cell 110:513–520

Rojas-Herrera R, Quiroz-Figueroa FR, Monforte-Gonzalez M, Sanchez-

Teyer F, Loyola-Vargas VM (2002) Differential gene expression

during somatic embryogenesis in Coffea arabica L., revealed by

RT-PCR differential display. Mol Biotechnol 21:43–50

Rupps A, Raschke J, Rummler M, Linke B, Zoglauer K (2016)

Identification of putative homologs of Larix decidua to

BABYBOOM (BBM), LEAFY COTYLEDON1 (LEC1),

WUSCHEL-related HOMEOBOX2 (WOX2) and SOMATIC

EMBRYOGENESIS RECEPTOR-like KINASE (SERK) during

somatic embryogenesis. Planta 243:473–488

Salaj J, Recklinghausen IR, Hecht V, de Vries SC, Schel JHN, van

Lammeren AAM (2008) AtSERK1 expression precedes and

coincides with early somatic embryogenesis in Arabidopsis

thaliana. Plant Physiol Biol 46:709–714

Santa-Catarina C, Hanai LR, Dornelas MC, Viana AM, Floh EIS

(2004) SERK gene homolog expression, polyamines and amino

acids associated with somatic embryogenic competence of

Ocotea catharinensis Mez. (Lauraceae). Plant Cell Tissue Organ

Cult 79:53–61

Santos D, Fevereiro P (2002) Loss of DNA methylation affects

somatic embryogenesis in Medicago truncatula. Plant Cell

Tissue Organ Cult 70:155–161

Santos MO, Romanoa E, Yotoko KSC, Tinoco MLP, Dias BBA,

Aragao FJL (2005) Characterization of the cacao somatic

embryogenesis receptor-like kinase (SERK) gene expressed

during somatic embryogenesis. Plant Sci 168:723–729

Savona M, Mattioli R, Nigro S, Falasca G, Della Rovere F,

Costantino P, De Vries S, Ruffoni B, Trovato M, Altamura

MM (2012) Two SERK genes are markers of pluripotency in

Cyclamen persicum Mill. J Exp Bot 63:471–488

Schellenbaum P, Mohler V, Wenzel G, Walter B (2008) Variation in

DNA methylation patterns of grapevine somaclones (Vitis

vinifera L.). BMC Plant Biol 8:78

Schlogl PS, Wendt dos Santos AL, Vieira LN, Floh EIS, Guerra MP

(2012a) Gene expression during early somatic embryogenesis in

Brazilian pine (Araucaria angustifolia (Bert) O. Ktze). Plant

Cell Tissue Organ Cult 108:173–180

Schlogl PS, Wendt dos Santos AL, Vieira LN, Floh EIS, Guerra MP

(2012b) Cloning and expression of embryogenesis-regulating

genes in Araucaria angustifolia (Bert.) O. Kuntze (Brazilian

Pine). Genet Mol Biol 35:172–181

Schmidt ED, Guzzo F, Toonen MA, de Vries SC (1997) A leucine-

rich repeat containing receptor-like kinase marks somatic plant

cells competent to form embryos. Development 124:2049–2062

Acta Physiol Plant (2017) 39:194 Page 15 of 17 194

123

Schmitt F, Oakeley EJ, Jost JP (1997) Antibiotics induce genome-

wide hypermethylation in cultured Nicotiana tabacum plants.

J Biol Chem 272:1534–1540

Schoof H, Lenhard M, Haecker A, Mayer KF, Jurgens G, Laux T

(2000) The stem cell population of Arabidopsis shoot meristems

is maintained by a regulatory loop between the CLAVATA and

WUSCHEL genes. Cell 100:635–644

Schrader S, Kaldenhoff R, Richter G (1997) Expression of novel

genes during somatic embryogenesis of suspension-cultured

carrot cells (Daucus carota). J Plant Physiol 50:63–68

Seefried WF, Willmann MRR, Clausen RL, Jenik PD (2014) Global

regulation of embryonic patterning in Arabidopsis by micro-

RNAs. Plant Physiol 165:670–687

Sharma S, Bryan G, Winfield M, Millam S (2007) Stability of potato

(Solanum tuberosum L.) plants regenerated via somatic embryos

axillary bud proliferated shoots, microtubers and true potato

seeds: a comparative phenotypic, cytogenetic and molecular

assessment. Planta 226:1449–1458

Sharma SK, Millam S, Hein I, Bryan GJ (2008) Cloning and

molecular characterization of a potato SERK gene transcription-

ally induced during initiation of somatic embryogenesis. Planta

228:319–330

Shen Y, Jiang Z, Yao X, Zhang Z, Lin H, Zhao M, Liu H, Peng H, Li

S, Pan G (2012) Genome expression profile analysis of the

immature maize embryo during dedifferentiation. PLoS One

7:e32237

Shen Y, Jiang Z, Lu S, Lin H, Gao S, Peng H, Yuan G, Liu L, Zhang

Z, Zhao M, Rong T, Pan G (2013) Combined small RNA and

degradome sequencing reveals microRNA regulation during

immature maize embryo dedifferentiation. Biochem Biophys

Res Commun 441:425–430

Shimada T, Hirabayashi T, Endo T, Fujii H, Kita M, Omura M (2005)

Isolation and characterization of the somatic embryogenesis

receptor-like kinase gene homologue (CitSERK1) from Citrus

unshiu Marc. Sci Hortic 103:233–238

Silva AT, Barduche D, Gabriella do Livramento K, Paiva LV (2015)

A putative BABY BOOM-like gene (CaBBM) is expressed in

embryogenic calli and embryogenic cell suspension culture of

Coffea arabica L. In Vitro Cell Dev Biol Plant 51:93–101

Singla B, Tyagi AK, Khurana JP, Khurana P (2007) Analysis of

expression profile of selected genes expressed during auxin-

induced somatic embryogenesis in leaf base system of wheat

(Triticum aestivum) and their possible interactions. Plant Mol

Biol 65:677–692

Sirohi G, Pandey BK, Deveshwar P, Giri J (2016) Emerging trends in

epigenetic regulation of nutrient deficiency response in plants.

Mol Biotechnol 58:159–171

Somleva MN, Schmidt EDL, de Vries SC (2000) Embryogenic cells

in Dactylis glomerata L. (Poaceae) explants identified by cell

tracking and by SERK expression. Plant Cell Rep 19:718–726

Stasolla C, Bozhkov PV, Chu TM, Van Zyl L, Egertsdotter U, Suarez

MF, Craig D, Wolfinger RD, Von Arnold S, Sederoff RR (2004)

Variation in transcript abundance during somatic embryogenesis

in gymnosperms. Tree Physiol 24:1073–1085

Stewards FC, Mapbs MO, Mears K (1958) Growth and organized

development of cultured cells. II. Organization in cultures grown

from freely suspended cells. Am J Bot 45:705–708

Stone SL, Braybrook SA, Paula SL, Kwong LW, Meuser J, Pelletier J,

Hsieh TF, Fischer RL, Goldberg RB, Harada JJ (2008)

Arabidopsis LEAFY COTYLEDON2 induces maturation traits

and auxin activity: implications for somatic embryogenesis. Proc

Natl Acad Sci USA 105:3151–3156

Su YH, Liu YB, Zhou C, Li XM, Zhang XS (2015) The

microRNA167 controls somatic embryogenesis in Arabidopsis

through regulating its target genes ARF6 and ARF8. Plant Cell

Tissue Organ Cult 124:405–417

Suer S, Agusti J, Sanchez P, Schwarz M, Greb T (2011) WOX4

imparts auxin responsiveness to cambium cells in Arabidopsis.

Plant Cell 23:3247–3259

Sunkar R, Girke T, Jain PK, Zhu JK (2005) Cloning and character-

ization of microRNAs from rice. Plant Cell 17:1397–1411

Thompson EW, Lane BG (1980) Relation of protein synthesis in

imbibing wheat embryos to the cell-free translational capacities

of bulk mRNA from dry and imbibing embryos. J Biol Chem

255:5965–5970

Tsuwamoto R, Yokoi S, Takahata Y (2010) Arabidopsis EMBRYO-

MAKER encoding an AP2domain transcription factor plays a

key role in developmental change from vegetative to embryonic

phase. Plant Mol Biol 73:481–492

Uddin MR (1993) Somatic embryogenesis in gymnosperms. US

5187092 A

Vashisht D, Nodine MD (2014) MicroRNA functions in plant

embryos. Biochem Soc Trans 42:352–357

Verbsky ML, Richards EJ (2001) Chromatin remodeling in Plants.

Curr Opin Plant Biol 4:494–500

Verdeil JL, Alemanno L, Niemenak N, Tranbarger TJ (2007)

Pluripotent versus totipotent plant stem cells: dependence versus

autonomy? Trends Plant Sci 12:243–252

Viejo M, Rodrıguez R, Valledor L, Perez M, Canal MJ, Hasbun R

(2010) DNA methylation during sexual embryogenesis and

implications on the induction of somatic embryogenesis in

Castanea sativa Miller. Sex Plant Rep 23:315–323

Waites R, Simon R (2000) Signaling cell fate in plant meristems:

three clubs on one tousle. Cell 103:835–838

Wang H, Caruso LV, Downie AB, Perry SE (2004) The embryo

MADS domain protein AGAMOUS-Like 15 directly regulates

expression of a gene encoding an enzyme involved in gibberellin

metabolism. Plant Cell 16:1206–1219

Wang X, Niu QW, Teng C, Li C, Mu J, Chua NH, Zuo J (2009)

Overexpression of PGA37/MYB118 and MYB115 promotes

vegetative-to-embryonic transition in Arabidopsis. Cell Res

19:224–235

Willemsen V, Scheres B (2004) Mechanisms of pattern formation in

plant embryogenesis. Annu Rev Genet 38:587–614

Williams EG, Maheswaran G (1986) Somatic embryogenesis: factors

influencing coordinated behaviour of cells as an embryogenic

group. Ann Bot 57:443–462

Williams L, Zhao J, Morozova N, Li Y, Avivi Y, Grafi G (2003)

Chromatin reorganization accompanying cellular dedifferentiation

is associated with modifications of histone H3, redistribution of

HP1, and activation of E2F-target genes. Dev Dyn 128:113–120

Willmann MR, Mehalick AJ, Packer RL, Jenik PD (2011) Micro-

RNAs regulate the timing of embryo maturation in Arabidopsis.

Plant Physiol 155:1871–1884

Wojcik AM, Gaj MD (2016) miR393 contributes to the embryogenic

transition induced in vitro in Arabidopsis via the modification of

the tissue sensitivity to auxin treatment. Planta 244:231–243

Wu XM, Liu MY, Ge XX, Xu Q, Guo WW (2011) Stage and tissue

specific modulation of ten conserved miRNAs and their targets

during somatic embryogenesis of Valencia sweet orange. Planta

233:495–505

Wurtele S, Wang H, Durgerian S, Nikolau BJ, Ulrich TH (1993)

Characterization of a gene that is expressed early in somatic

embryogenesis of Daucus carota. Plant Physiol 102:303–312

Xu M, Li X, Korban S (2004) DNA-methylation alterations and

exchanges during in vitro cellular differentiation in rose (Rosa

hybrida L.). Theor Appl Genet 109:899–910

Yang X, Zhang X (2010) Regulation of somatic embryogenesis in

higher plants. Crit Rev Plant Sci 29:36–57

Yang X, Wang L, Yuan D, Lindsey K, Zhang X (2013) Small RNA

and degradome sequencing reveal complex miRNA regulation

during cotton somatic embryogenesis. J Exp Bot 64:1521–1536

194 Page 16 of 17 Acta Physiol Plant (2017) 39:194

123

Yeung EC (1995) Structural and development patterns in somatic

embryogenesis. In: Thorpe TA (ed) In vitro embryogenesis in

plants. Kluwer, Netherlands, pp 205–247

Zhai L, Xu L, Wang Y, Zhu X, Feng H, Li C, Luo X, Everlyne MM,

Liu L (2016) Transcriptional identification and characterization

of differentially expressed genes associated with embryogenesis

in radish (Raphanus sativus L). Sci Rep 6:21652

Zhang C, Hsieh TF (2013) Heritable epigenetic variation and its

potential applications for crop improvement. Plant Breed

Biotechnol 1:307–319

Zhang S, Liu X, Lin Y, Xie G, Fu F, Liu H, Wang J, Gao S, Lan H,

Rong T (2011) Characterization of a ZmSERK gene and its

relationship to somatic embryogenesis in a maize culture. Plant

Cell Tissue Organ Cult 105:29–37

Zhang C, Cao L, Rong L, An Z, Zhou W, Ma J, Shen WH, Zhu Y,

Dong A (2015a) The chromatin-remodeling factor AtINO80

plays crucial roles in genome stability maintenance and in plant

development. Plant J 82:655–668

Zhang F, Dong W, Huang L, Song A, Wang H, Fang W, Chen F,

Teng N (2015b) Identification of microRNAs and their targets

associated with embryo abortion during Chrysanthemum cross

breeding via high-throughput sequencing. PLoS One

10:e0124371

Zheng Y, Ren N, Wang H, Stromberg AJ, Perry SE (2009) Global

identification of targets of the Arabidopsis MADS domain

protein AGAMOUS-Like15. Plant Cell 21:2563–2577

Zheng Q, Zheng Y, Perry SE (2013) AGAMOUS-Like15 promotes

somatic embryogenesis in Arabidopsis and Soybean in part by

the control of ethylene biosynthesis and response. Plant Physiol

161:2113–2127

Zheng W, Zhang X, Yang Z, Wu J, Li F, Duan L, Liu C, Lu L, Zhang

C, Li F (2014) AtWuschel promotes formation of the embryo-

genic callus in Gossypium hirsutum. PLoS One 9:e87502

Zhu QH, Shan WX, Ayliffe MA, Wang MB (2016) Epigenetic

mechanisms: an emerging player in plant-microbe interactions.

Mol Plant Microbe Interact 29:187–196

Zuo J, Niu QW, Frugis G, Chua NH (2002) The WUSCHEL gene

promotes vegetative-to-embryonic transition in Arabidopsis.

Plant J 30:349–359

Acta Physiol Plant (2017) 39:194 Page 17 of 17 194

123

本文献由“学霸图书馆-文献云下载”收集自网络，仅供学习交流使用。

学霸图书馆（www.xuebalib.com）是一个“整合众多图书馆数据库资源，

提供一站式文献检索和下载服务”的24 小时在线不限IP

图书馆。

图书馆致力于便利、促进学习与科研，提供最强文献下载服务。

图书馆导航：

图书馆首页 文献云下载 图书馆入口 外文数据库大全 疑难文献辅助工具