Duarte-Aké 2016

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Chapter 6
Epigenetic Advances in Somatic
Embryogenesis in Sequenced
Genome Crops
Fátima Duarte-Akéand Clelia De-la-Peña
Abstract Despite the promise of genetic sequencing for the field, more than 100
sequenced plants so far have not shed light on the biological process of somatic
embryogenesis. Today it is known that there are differences between SE and its
counterpart, zygotic embryogenesis (ZE). SE is impossible to induce in some
plants, while others have very reproducible protocols. Advances in molecular
biology and the biochemistry behind the SE process indicate that plants share some
of the SE-related genes and regulatory pathways. However, the primary difference
in response is in the sensing of plant growth regulators. There are plants that need to
be exposed to auxins to initiate the SE process, while others do not need any auxin
at all. Either way, once the induction has started, gene regulation follows the course
and new embryogenic structures start to emerge. One important component of gene
regulation is epigenetic modifications, such as DNA methylation and histone
modifications. These mechanisms have been studied in different plants, such as
monocots and dicots, and differences have been linked to class. In this chapter we
will discuss the work done in SE in different sequenced crops of agronomic
importance, including sugarcane, maize, coffee, orange, cacao and others.
6.1 Introduction
Somatic embryogenesis (SE) is the process by which a single somatic cell or a
group of somatic cells originate a somatic embryo that develops in several distinct
stages. These stages are labeled globular, heart-shaped and cotyledon-shaped, in
cotyledon species; globular scutellar and coleoptilar stages in monocots; and
globular, early cotyledonary and late cotyledonary embryos in conifers. SE is an
important tool for clonal propagation of important economical and agronomical
species (Loyola-Vargas and Ochoa-Alejo 2012). The success of this technique lies
F. Duarte-AkéC. De-la-Peña(&)
Centro de Investigación Científica de Yucatán, Unidad Biotecnología,
Calle 43 no. 130, Col. Chuburnáde Hidalgo, CP 97200 Mérida, Yucatán, Mexico
e-mail: clelia@cicy.mx
©Springer International Publishing Switzerland 2016
V.M. Loyola-Vargas and N. Ochoa-Alejo (eds.), Somatic Embryogenesis:
Fundamental Aspects and Applications, DOI 10.1007/978-3-319-33705-0_6
81

in the high number of embryos produced for explant, which can be converted into
functional plants. The switching of development that suffers a somatic cell to
regenerate to a complete plant involves several events associated with molecular
recognition of internal and external signals (Nic-Can et al. 2015,b; Joshi et al.
2013). This eventually results in specific gene expression of different families of
genes that are involved in plant growth perception, endogenous homeostasis of
growth regulators, transduction signal and morphogenic response (Yang and Zhang
2010).
Environmental factors and the conditions of the growth culture are inherent
factors that control the interactions among epigenetic mechanisms during SE
(Nic-Can et al. 2015; Nic-Can and De-la-Peña2014; De-la-Peña et al. 2015). The
epigenetic mechanisms, such as DNA methylation, post-transduction covalent
modifications of histones, and small RNAs, act together to regulate gene expression
(Wang et al. 2015; Us-Camas et al. 2014; Allis et al. 2015).
We are close to understanding the role of epigenetic mechanisms during SE in
different model plants; however, not all findings can be applied to non-model
plants, even within the same genus (Nic-Can et al. 2015), or to plants with different
metabolisms (Duarte-Akéet al. unpublished data). In this chapter, we discuss the
work done in SE to apply epigenetic understanding to sequenced plants of agro-
nomic relevance, such as sugarcane, maize, coffee, orange, cacao, beans and others.
6.2 Genes Involved in Somatic Embryogenesis
In plant biology, zygotic embryogenesis (ZE) is a model to study the expression of
genes and translated proteins in the signal response during embryo development.
However, SE has been found to be a useful tool as well, and can probe different
questions surrounding these phenomena, and in a more controlled way. Due to the
advantages of SE for the culturing of thousands of crop plants of economical
importance, a field of study has been opened to understand the regulatory processes
involved during the initiation and progress of different embryo developmental
stages. The most studied genes regarding these processes are listed below.
6.2.1 SERK1
SOMATIC EMBRYOGENESIS RECEPTOR-LIKE KINASE (SERK) is a trans-
membrane receptor that has been implicated in triggering embryogenesis. It was
isolated for the first time from Daucus carota suspension cells, where expression
was up regulated in the globular stage of embryogenesis (Schmidt et al. 1997).
SERK is activated via autophosphorylation, specifically in the residue threonine
468 (Shah et al. 2001), glutathione S-transferase fusion and in vitro phosphorylation
assays. SERK traduces the signal from cell membrane to action site, regulating the
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subsequent downstream proteins in the signal transduction pathway. To date, dif-
ferent experiments suggest the pivotal role of this protein in the initiation of
embryogenic competence. For instance, the ortholog SERK of Arabidopsis
enhances the ability to undergo the somatic embryo response (Hecht et al. 2001).
The histochemical and immunochemical techniques using β-glucuronidase detec-
tion have revealed the spatial localization of gene expression during indirect
somatic embryogenesis. The expression was detected in four places: in embryo-
genic callus, where the embryos were developed, in the basal parts of the embryos,
in the outer layers of cotyledons and in the provascular and vascular strands of
developing somatic embryos.
6.2.2 LEC
The LEAFY COTYLEDON (LEC) genes are classified into two classes. The first
class are HAP3- related transcription factors, represented in Arabidopsis by LEC1
and L1L (Lotan et al. 1998; Kwong et al. 2003). The second class encodes B3
domain transcription factors, which are represented by LEC2, FUS3, and ABI3 in
Arabidopsis (Stone et al. 2001,2008;Mönke et al. 2012; Luerßen et al. 1998).
Among the LEC genes, LEC1 yLEC2 have been suggested to have complementary
and partial redundancy to induce SE. They have similar but not identical functions,
as lec1lec2 double mutants have synergistic phenotypes (West et al. 1994; Lotan
et al. 1998; Stone et al. 2001). Conversely, the overexpressing phenotypes of LEC1
seedlings arrest embryo-like seedlings and fail to develop; cotyledon-like organs
sometimes form in place of the first leaves (Lotan et al. 1998). LEC1 has been
associated with the maturation of embryos because in LEC1 mutants the phenotype
is defective and the conversion from cotyledons to leaf-like structures does not take
place. In LEC2, the embryo-like seedlings continued to proliferate, producing
callus, cotyledon-like structures and leaf-like organs in addition to the SE.
Transgenic plants expressing LEC2 ectopically develop somatic embryos, which
have cotyledon-like and leaf-like structures (Stone et al. 2001).
6.2.3 AGL15
AGAMOUS-LIKE15 (AGL15) belongs to the MAD-box proteins family, which is
involved in the control of flowering time, homeotic regulation of floral organo-
genesis, fruit formation and seed pigmentation (Parenicováet al. 2003; Rounsley
et al. 1995). The role of AGL15 during embryo development was studied in
Arabidopsis and Brassica napus. In Brassica embryos, embryonic tissues, and early
and late globular stages, a great accumulation of mRNA of AGL15 was demon-
strated by in situ hybridization techniques, suggesting a regulatory role during
embryo development. In other species the presence of AGL15 has been detected
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during embryo development. For instance, in the soybean, GmAGL15 was present
in young embryos and the highest expression was detectable in somatic embryo
cultures (Thakare et al. 2008). Furthermore, this transcription factor promotes the
development of somatic embryos and enhances embryonic tissue production in
Arabidopsis (Harding et al. 2003).
6.2.4 BBM
BABY BOOM (BBM), which is a member of the APETALA2-Ethylen responsive
factor family (AP2-ERF), is a transcription factor involved in cell proliferation and
morphogenesis during embryogenesis. BBM was isolated from an androgenic
system in B. napus, where it is expressed during seed and embryo development
(Boutilier et al. 2002). When this gene is overexpressed ectopically in Arabidopsis
and B. napus, it can promote somatic embryos. The ectopic expression of BBM
leads to spontaneous embryo formation from seedlings, as well as ectopic shoots
and calli at lower frequency (Boutilier et al. 2002). In tobacco, the heterologous
expression of BBM promotes the formation of both callus and shoots (Srinivasan
et al. 2007).
6.2.5 CLV
There are three CLAVATA (CLV) genes that are important for meristem develop-
ment; these are CLV1,CLV2 and CLV3 (Nikolaev et al. 2007). CLV1, which
encodes to a transmembrane receptor kinase, promotes the progression of meristem
cells toward organ initiation throughout a signal cascade for the initiation of
organogenesis (Clark et al. 1996,1997). CLV2 encodes to a receptor-like protein
and is required for the accumulation of CLV1 with which to form a heterodimer
useful for signaling transduction (Jeong et al. 1999). In the case of CLV3, this gene
is expressed in the central zone cells of the shoot apical meristems and is a key
regulator for shoot and meristem development (Clark et al. 1995).
6.2.6 WUS
WUSCHELL (WUS) encodes to a transcription factor that directly regulates other
genes (Lenhard and Laux 1999). WUS is a homeodomain protein that promotes SE
when it is ectopically expressed (Zuo et al. 2002). Its principal function is to
maintain the undifferentiated state of cells and in response to different stimuli to
switch the developmental fate of tissues (Gallois et al. 2004).
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During somatic embryogenesis of Picea abies, a member of the WUS genes,
PaWOX2, is highly expressed in the early stage and its expression gradually
declines when the embryos mature. PaWOX2 expression can be used as a marker
for embryogenic potential in embryogenic cell cultures (Palovaara and Hakman
2008). Furthermore, the expression of WUS depends on other factors such as
proteins (e.g., CLAVATA) and growth regulators (e.g., auxins). For example, in
Medicago truncatula, the overexpression of WOX5 provokes a highly embryogenic
callus formation when the explant is cultured in the presence of auxin, whereas
without auxin in the medium, the embryo formation is direct. In both cases the
expression of WOX genes stimulates embryo formation in the presence and absence
of auxin (Imin et al. 2007).
6.2.7 GH3
GRETCHEN HAGEN 3 (GH3) genes, found in many plants (Terol et al. 2006),
were discovered in Glycine max in response to auxins (Hagen et al. 1984).
Evolutionary data show that GH3 proteins descend from a common ancestral
chromosome before the eudicot/monocot splits (Okrent and Wildermuth 2011).
GH3 genes have important roles in the regulation of the stress response in
Arabidopsis (Park et al. 2007) and have been classified into three main groups
(Chen et al. 2010). Group 1 contains AtGH3.11 (JAR1/FIN219), which adenylates
jasmonic acid (JA), and AtGH3.10 (DFL2) (Staswick and Tiryaki 2004). On the
other hand, AtGH3.2 (YDK), AtGH3.5 (AtGH3a), AtGH3.6 (DFL1), and
AtGH3.17, which belong to group 2, adenylate indolacetic acid (IAA) and conju-
gate with salicylic acid (SA) (Staswick et al. 2005,2002). Furthermore, AtGH3.5,
AtGH3.6 and AtGH3.17 are suggested to be targets of the auxin response factor 8
(ARF8) (Tian et al. 2004). Group 3 of the GH3 genes contains AtGH3.9, AtGH3.12
and AtGH3.17; both AtGH3.9 and AtGH3.17 are activated by IAA (Okrent and
Wildermuth 2011).
6.2.8 ARF
AUXIN RESPONSE FACTOR (ARF) encodes a transcription factor that activates
minutes after auxin stimulation (Ulmasov et al. 1999; Smit and Weijers 2015).
There are 23 ARF genes in Arabidopsis and they have different functions
(Okushima et al. 2005; Remington et al. 2004; Guilfoyle and Hagen 2007; Smit and
Weijers 2015; Hamann et al. 2002; Weijers et al. 2006; Weijers and Jürgens 2005).
For instance, ARF 1 and 3are involved in fruit development (de Jong et al. 2009;
Guillon et al. 2008; Goetz et al. 2007); ARF2 in senescence (Lim et al. 2010); ARF
5,17 and 23 in embryogenesis (Weijers et al. 2006; Hamann et al. 2002); and ARF
6and 8in the expansion of leaves (Nagpal et al. 2005; Tian et al. 2004). Moreover,
6 Epigenetic Advances in Somatic Embryogenesis …85

ARF 7,10,16 and 19 have been shown to be involved in lateral root formation and
development (Marin et al. 2010; Okushima et al. 2007; Tatematsu et al. 2004),
while ARF 12,13,14,20,21 and 22 respond to other plant hormones such as
ethylene (Li et al. 2006), brassino steroids (Vert et al. 2008) and abscisic acid
(Yoon et al. 2010; Xie et al. 2015).
6.2.9 PIN1
PIN-FORMED (PIN) proteins are involved in the polar auxin transport across cell
membranes (Petrášek et al. 2006;Petrášek and Friml 2009;Křeček et al. 2009). In
Arabidopsis, the PIN family is formed by eight genes, which are divided in two
subclades. In the first subclade there are the canonical PINs, consisting of PIN1-4
and PIN7. These are localized in the plasma membrane and function as auxin efflux
transporters in a direct manner. The second subclade consisting of PIN5,PIN6 and
PIN8, which are localized in the endomembranes, suggesting their role in auxin
distribution and homeostasis in the intracellular compartments (Rodríguez-Sanz
et al. 2015). Among the canonical PINs, PIN1 is the protein that has a central role
during embryogenesis. PIN1 controls the direction of polar auxin transport in
embryo development and the establishment of polarized auxin fluxes, auxin gra-
dients and auxin maxima in the basal and apical regions at defined developmental
embryo stages. This protein is accumulated during the earliest developmental stages
in the cells that would become embryos. That accumulation moves from nonpolar
to polarized and then to the basal side of the provascular cells once the early
globular stage is reached (Friml et al. 2003). In addition to PIN1,PIN4 is also
expressed in the proembryo at the globular stages and is functionally redundant to
PIN1 in the seedling stage (Vieten et al. 2005; Weijers et al. 2006).
6.2.10 LEA
LATE EMBRYOGENESIS ABUNDANT (LEA) genes are expressed only in the later
stages of embryo maturation, a fact which has been used as a molecular marker to
discriminate between direct and indirect somatic embryogenesis (Corre et al. 1996).
6.2.11 STM
The genes SHOOTMERISTEMLESS (STM) and CLAVATA 1 (CLV1) were isolated
from Brassica species, and ectopically expressed in Arabidopsis to understand the
embryogenic process (Elhiti et al. 2010). The ectopic expression of these orthol-
ogous genes affects embryo production in vitro. The ectopic expression of BoSTM,
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BrSTM and BnSTM increased the number of somatic embryos obtained, whereas in
ectopic expression of CLV1, the embryo yield was repressed. Therefore, the
antagonistic expression of these genes is necessary for SE coordination.
6.3 Epigenetic Studies on Sequenced Plants
The sequencing of plant crop genomes is a helpful tool to understand the process of
SE, and when these genomes are shared in public databases, gene sequence searches
become effective ways to analyze differential regulation. The massive sequencing
methods applied to generate genome databases of economically important species
have helped us to understand the evolutionary aspects of different genes among
plants, as well as the loss or gain of certain genes or groups of genes. Although
transcriptomics allows the identification of important genes that are up or down
regulated in response to different environmental conditions, the mechanism of
regulation is unknown at this level.
The study of epigenetics contributes a new level of understanding of the regu-
lation of gene expression throughout chromatin conformation. DNA methylation,
histone post-translational modifications and the microRNA (miRNA) mechanism
are now enabling us to uncover gene regulation during the embryogenic response. It
is known that SE is difficult to attain in some species due to genetic or epigenetic
variability (Schaffer 1990; Miguel and Marum 2011; Phillips et al. 1994; Kaeppler
et al. 2000; De-la-Peña et al. 2015). The micropropagation of small fruits such as
cranberry and blueberries, as well as many non-model plants, traditionally prob-
lematic. However, the studies listed below have shown that epigenetic regulation
can be used in order to improve the SE response. Here we describe studies on
epigenetic and SE done in some important sequenced genome species of agro-
nomical importance. Although almost 100 plants have been sequenced already, few
have been the species that are propagated by SE and fewer yet are those for which
epigenetic mechanisms have been analyzed.
6.3.1 Barley
Hordeum vulgare L. is one of the world´s earliest domesticated crop species and
represents the fourth most abundant cereal (http://faostat.fao.org). Barley is resistant
to different environmental conditions and for this reason is cultivated and consumed
in arid and marginal regions. The improvement and scale production of this crop is
performed by in vitro systems (Jähne et al. 1991;Lührs and Lörz 1987). In partic-
ular, microspore embryogenesis is an important tool in breeding to obtain
double-haploid plants (Mordhorst and Lörz 1993; Solís et al. 2015). During this
process, changes in differentiation and proliferation are regulated by DNA methy-
lation (Solís et al. 2015). The study of the DNA methylation dynamic using 5-Aza
6 Epigenetic Advances in Somatic Embryogenesis …87

have revealed that this drug, at a concentration of 2.5 μm for 4 days, can induce a
major embryo production as a consequence of the DNA hypomethylation. In con-
trast, the longer the treatment with 5-Aza, the lower the embryo production becomes.
These results suggest a key role of DNA methylation in totipotency acquisition and
microspore reprograming in barley. This can be used as a powerful tool to improve
embryo production not only in barley but in other important crops as well.
6.3.2 Common Bean
Phaseolus vulgaris L., the common bean, is the most consumed legume in México.
This species is recalcitrant to both SE and in vitro regeneration. For this reason, the
study of SE in the induction process is necessary to understand the process and
improve the protocols in order to achieve a better regeneration rate. For instance,
Barraza et al. (2015) have used a regeneration-competent callus that was succes-
sively transformed. These embryogenic calli were regenerated and transformed with
a PvTRX1hRiA construction to down regulate the expression of the PvTRX1h gene.
This gene is an ortholog of a lysine methyltransferase in plants. The low expression
of PvTRX1h not only altered the concentration of the hormone content in the calli
but also affected the expression of important genes involved in auxin biosynthesis.
On the other hand, the down regulation of PvTRX1h activated the expression of
other histone lysine methyltransferases, such as PvASHH2h. These results suggest
a crosstalk among histone methyltransferases, with plant regulators signaling for the
generation of somatic embryos.
6.3.3 Brassica
In the STM overexpression line of Brassica oleracea the expression of genes
involved in hormone perception and signaling, as well as genes encoding DNA
methyltransferases, were affected (Elhiti et al. 2010). On the other hand, pharma-
cological experiments performed to confirm some of these results showed that
Arabidopsis SE is encouraged by a global DNA hypomethylation during the
induction (when in presence of 2, 4-D, the cells acquire the competency to form
embryonic cells) (Elhiti et al. 2010).
6.3.4 Cacao
Theobroma cacao L. is endemic to South American rainforests and was domesti-
cated approximately three hundred years ago in Central America. This species is a
very important tree crop because it is the source of chocolate. One of the main
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problems for chocolate production is that the plant is susceptible to many pests and
diseases (Iwaro et al. 2006) and the difficult cultivation via apical microcutting for
large-scale production has led to an increasing interest in the application of SE for
clonal multiplication (Traore et al. 2003). However, cocoa SE carries an elevated
risk for genetic mutations, and as a result the genetic and epigenetic variation has
been evaluated in this species (Rodríguez López et al. 2010). The simple sequence
repeat (SSR) markers and methyl-sensitive amplification polymorphism (MSAP)
analysis have revealed high genetic and epigenetic variation, respectively. In the
somatic callus was found a possible interaction of DNA methylation with aberrant
recombinant events during the embryogenesis, which might allow de novo mutation
(Rodríguez López et al. 2010).
6.3.5 Coffee
Every day more than 2.25 billion cups of coffee are consumed around the world.
Approximately 11 million hectares are cultivated to supply this demand for coffee
(Denoeud et al. 2014). There are two economically important species of coffee:
Coffea arabica and Coffea canephora, which represent 70 and 30 % of the coffee
produced in the world, respectively (Mondego et al. 2011). In coffee production, SE
is applied industrially for large-scale and rapid dissemination of selected hybrids
that provide an increase in the yield of high quality coffee, and promises an efficient
system for the multiplication of varieties with modified caffeine content (Etienne
et al. 2012; Bertrand et al. 2011; Ogita et al. 2003). Studies on the epigenetics in
coffee have attempted to understand the embryogenic capacity (Nic-Can et al. 2015,
2013b) and the somaclonal variations present in coffee multiplication (Bobadilla
Landey et al. 2015,2013).
In C. canephora, treatment with 5-azacitidine (5-Aza, an inhibitor of DNA
methylation) revealed that DNA methylation is important for embryo development
by disturbing the expression of important genes involved in SE, such as LEC1 and
BBM1 (Nic-Can et al. 2013b). Another recent study (Nic-Can et al. 2015) shows
that the treatment of C. canephora explants of low molecular mass with the con-
ditioned medium fraction from C. arabica not only reduced the number of embryos
per explant but also affected the DNA methylation levels, thus revealing the
importance of DNA methylation during embryogenic competence for embryo
formation. In coffee, SE has also indicated the importance of histone
post-translational modifications (Nic-Can et al. 2013a,b). In C. canephora, the
coordinated expression of genes involved in SE, such as WOX4,LEC1 and BBM1,
is regulated by the epigenetic marks H3K9me2 and H3K27me3.
6 Epigenetic Advances in Somatic Embryogenesis …89

6.3.6 Cotton
Gossypium hirsutum is the most important natural textile fiber and its seed is an
important source of feed and foodstuff. Cotton breeding is dependent on in vitro
systems for scale production (Kim and Triplett 2001). SE is an effective plant
regeneration procedure from transgenic cotton propagation (Zhang et al. 2001).
Cotton SE is a process that provides an outstanding experimental tool for studying
the biochemical and molecular bases of cellular SE in recalcitrant genotypes.
In cotton SE studies there have been identified two phases of chromatin reor-
ganization associated with endogenous auxin/cytokinin dynamic activity that may
underlie dedifferentiation and redifferentiation events (Zeng et al. 2007). On the
other hand, in studies on miRNAs during the SE of cotton, it was found that 36
differentially expressed known miRNA families and 25 novel microRNAs with 476
genes as targets were involved in the process (Yang et al. 2013). The expression
patterns of miRNAs and their targets were validated. For instance, the expression of
miRNA 167 and 156 were evaluated and correlated with the expression of the target
genes ARF and SPL, respectively.
6.3.7 Eggplant
Solanum melongena L. is a vegetable crop species, which is genetically important
due to its different agronomical qualities, such as extra-large fruit size, high tolerance
to biotic and abiotic stresses, and parthenocarpy without any negative pleiotropic
effects. These are characteristics that would help to improve other important sola-
naceum species. Highly effective protocols for in vitro plant regeneration via SE
from cotyledon explants are available in eggplant to understand the organogenic and
embryogenic process. In the embryogenic process, the changes regulated by epi-
genetic mechanisms are necessary for embryo generation. One of the most studied
mechanisms is DNA methylation. Using isoschizomer restriction enzymes MspI and
HpaII, it was found that the methylation varied widely in the DNA (Bucherna et al.
2001). The sequences that show major changes in DNA methylation levels were in a
sequence that has a 91 % similarity to an uncharacterized sequence found in tomato
ovaries, so this transcript could have a function in morphogenesis and differentiation.
The authors propose that this epigenetic mechanism may play a role in the regulation
of gene activity and cell differentiation in embryogenic suspensions.
6.3.8 Grapevine
Vitis vinifera L. is one of the most important cultivars for both fruit and beverage
production. Due to the limited natural variability of the cultivars, traditional vine
improvement is considered difficult. However, the in vitro embryogenic system is
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used as to select improved grapevine phenotypes with specific characteristics, such
as vigor, berry size, sugar and acid concentration, and flavor components. The effect
of in vitro embryogenesis in the grapevine genotypes was studied in two variants,
Chardonnay 96 and Syrah 174 (Schellenbaum et al. 2008). The study reveals that
the in vitro conditions cause a few changes in the polymorphism bands. However,
the somaclones can conserve their main characteristics, which is a prerequisite for
grapevine cultivation. Comparative MSAP analysis between mother plants and
somaclones has revealed that the somaclones have slightly higher methylation
levels in comparison with mother plants (Baranek et al. 2010).
6.3.9 Maize
Zea mays L. is one of the most important cultivated cereal crops and is used as
source of food, livestock feed and raw material for the industry (Huang et al. 2002).
Due to its relevant economic importance, maize is a great model for improvement.
In maize, the induction of SE starts in a specific balance of environmental condi-
tions, including darkness and growth regulators in the media (Conger et al. 1987).
Recently it was found that the induction process in maize is characterized by an
enrichment of small RNA molecules that are involved with SE initiation
(Chávez-Hernández et al. 2015). Chávez-Hernández et al. (2015) investigated the
accumulation of certain miRNAs, and their predicted targets, during the SE of maize
under different environmental conditions. It was found that miRNAs 156, 164, 168,
397, 398, 408, and 528 increased upon growth regulator depletion, while photoperiod
conditions increase the expression of the targets SBP23,GA-MYB,CUC2,AGO1c,
LAC2,SOD9,GR1,SOD1A and PLC. These results demonstrate that the concen-
tration of growth regulators has an influence on specific miRNA accumulation during
SE, while their targets are additionally influenced by the presence of light.
6.3.10 Medicago
The role of DNA methylation in the somatic embryogenesis of legumes was studied
using the model plant M. truncatula. In a pharmacological study, it was found that
the treatment with a Hypermethylation drought 5-Aza in a high embryogenic line
provokes a loss of embryogenic capacity, suggesting an important role for DNA
methylation in embryogenic capacity (Santos and Fevereiro 2002).
6.3.11 Norway Spruce
Picea abies (L.) Karst is one of the most widespread and ecologically and eco-
nomically important plants in Europe. In P. abies, SE protocols are used to
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understand the zygotic process, which is dependent on temperature and regulation
by epigenetic mechanisms. For instance, Yakovlev et al. (2014) identify differen-
tially expressed transcripts during the SE of Norway spruce. Their study revealed
that there is an epigenetic memory affected by temperature during the embryoge-
nesis process. It was found that the formation of the epigenetic memory induced by
warm and cold conditions during SE is accompanied by differential expression of
different sets of genes. These included epigenetic machinery-related genes, such as
DNA methyltransferases, histone methyltransferases, histone acetylases and histone
deacetylases.
6.3.12 Oak
Quercus suber L. is an ecologically and economically important species cultivated
in the Mediterranean area. The in vitro culture of Q. suber L. via SE is an alter-
native to conventional methods of reproduction that solve the problem of its long
reproductive propagation and irregular seed yield. However, the major bottleneck
for its micropropagation is the complete maturation of the embryos. It is known that
SE is an event controlled by epigenetic mechanisms additional to the genetic and
biochemical regulation. Recently, it was discovered that genes necessary for
embryo maturation are under the regulation of histone H3 modifications and
chromatin remodeling (Pérez et al. 2015a).
SE in oak is a complex process, where, in addition to epigenetic controls,
hormonal pressure plays an important role in embryo maturation (Pérez et al.
2015b). A biochemical and immunohistochemical analysis has revealed that the
beginning of embryo maturation is characterized by a peak in the ABA levels, while
the acquisition of germinative capacity needs a cold treatment. The germination
ability is accompanied by a decrease in ABA levels as well as DNA methylation
status in the meristematic areas. This work opens the possibility of using
demethylating agents or ABA inhibitors to improve the number and quality of the
mature somatic embryos, reducing the time of cultures.
6.3.13 Oil Palm
Elaeis guineensis Jacq., the most efficient African oil palm, has a long life cycle,
around 25 years. Because this plant is recalcitrant to vegetative propagation,
in vitro propagation methods based on SE have been implemented (Pannetier et al.
1981). However, around 5 % of somatic embryo-derived palms show abnormalities
in their floral development, causing a mantled phenotype, which is the feminization
of the male parts in flowers of both sexes. This somaclonal variation was observed
in different proportion between two types of calli. Five percent of the regenerants
from nodular compact calli (NCC) presented mantled phenotype while the fast
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growing calli (FGC) produced 100 % of the variant palms. The two types of calli
that were used have the same genotype and they were cultured in same conditions.
Therefore, this variation was found to be of an epigenetic nature (Jaligot et al.
2000). The epigenetic mechanism involved in this somaclonal variation was
recently identified (Ong-Abdullah et al. 2015). The plants with abnormal pheno-
types are hypomethylated near the Karma transposon splice site, which prevents the
correct transcription of the homeotic gene DEFICIENS, which is involved in the
correct development of the floral parts. This situation was due to embryogenic
culturing; however, the discovery of the epigenetic mechanism involved in this
somaclonal variation would facilitate the introduction of higher-performing clones
and optimize the yield of oil palm production.
6.3.14 Rice
Rice (Oriza sativa L.) is one of the most important crop plants of the world, feeding
over half of the global population. Due to its economic importance, rice is con-
sidered to be a model plant for studies on genomic and epigenetic research. In rice,
the analysis of abundances and identification of the differential miRNAs between
the non-differentiated state and the differentiated calli has revealed that miRNA 397
is more abundant in the non-differentiated calli, while 156 is found at high levels in
the differentiated tissue (Luo et al. 2006). MiRNA 397 has been involved in mature
tissues and targeted LACCASE genes, which might be vital to maintain the
meristematic state in non-differentiated callus. Comparing the sequenced miRNAs
between undifferentiated and differentiated calli, it was noticed that around 50
miRNA sequences vary in abundance between the two types of calli, suggesting a
differential role in meristem development. Among them, the miRNA 408, 397 and
528, which are strongly expressed in rice seeds, have a significantly higher abun-
dance in undifferentiated calli, in contrast with the differentiated calli. These data
suggest that miRNAs would modulate the development of meristems by regulating
the expression of crucial target genes involved in the differentiation process (Chen
et al. 2011).
6.3.15 Rubber Tree
Havea brasiliensis Willd. is the major commercial source of natural rubber, a latex
polymer with high elasticity, flexibility and resilience that is used in the manu-
facture of over 50,000 products (Nair 2010). The high variation in the plantation of
rubber trees is a common problem due to the use of bud grafting for the propagation
of the rootstocks (Carron et al. 2009; Omokhafe 2004). Although SE studies have
not been performed in this plant, the use of zygotic cleavage polyembryony in
immature fruits opens a new avenue for the successful multiplication of rubber
6 Epigenetic Advances in Somatic Embryogenesis …93

trees. Epigenetic analysis was performed on different samples to determine global
DNA methylation using MSAP. However, under these induction conditions no
epigenetic variation was found (Karumamkandathil et al. 2015).
6.3.16 Sugar Beet
Beta vulgaris is an important crop of temperate climates. This plant is cultivated on
two million hectares worldwide. Sugar beet provides nearly 30 % of the world´s
annual sugar production and it is a source for bioethanol and animal feed. Its
genome was sequenced in 2014, opening the opportunity for breeding and man-
agement of different epigenetic aspects associated with development of this eco-
nomically important crop (Dohm et al. 2014).
This species is propagated by different protocols of embryogenesis for both
multiplication and breeding (Zhang et al. 2008). Among the unexplored elements in
the breeding of this plant is the epigenetic changes that occur during in vitro
propagation (Kornienko et al. 2014). For instance, in sugar beet, acquisition of
competence to generate embryos from an explant, and the subsequent proliferation
and morphogenesis events, are dependent on changes in DNA methylation levels.
On the other hand, in this species the dynamic connection between plant mor-
phogenesis, cell redox state and the changes in DNA methylation and H3 acety-
lation marks was evident (Causevic et al. 2005,2006). Causevic et al. (2006)
demonstrated that the distinct morphogenic capacity of tree lines, organogenic (O),
non organogenic (NO) and dedifferentiated (DD), are associated with different
levels of epigenetic parameters and corresponding enzymes, such as DNMT and
HDAC, which catalyze DNA methylation and histone acetylation, respectively.
The DD line presents more reactive oxygen species (ROS) and nonenzymatic
antioxidant properties. Additionally, this line presented hypermethylation in a key
enzyme that is fundamental to oxidative stress, such as the super oxide dismutase
(SOD). In the case of the lines O and NO, the level of acetylation in H3 was
reduced.
6.3.17 Valencia Sweet Orange
Citrus fruits and juice are the prime human source for vitamin C, an important
component of human nutrition. In the aim of improving citrus crops, different
biotechnological approaches such as transformation, protoplast fusion, in vitro
mutation breeding and SE have been used with success for the regeneration of
plants of Citrus sinensis (Xu et al. 2013; Gmitter et al. 2012).
Most of the epigenetic studies on SE have been on DNA methylation or histone
modifications. However, in the case of SE in citrus, the small RNA has been
extensively studied. The abundance and presence of certain micro RNAs (miRNAs)
94 F. Duarte-Akéand C. De-la-Peña

and the effect on predicted targets have contributed to the understanding of plant
cell totipotency as well as embryogenic capacity of somatic cells. For instance, in C.
sinensis around ten conserved plant miRNAs were detected and sixteen genes were
predicted to be targeted by six miRNAs (Wu et al. 2011). Stage- and tissue-specific
expressions of miRNAs and their targets suggest the involvement of these small
molecules in the modulation of SE. During the induction process, the abundance of
miRNA156, 168 and 171 is necessary for embryogenic competence and for embryo
formation. In non-embryogenic callus, these microRNAs are not present, or their
abundance is low (Wu et al. 2011). Interestingly, miRNA156 has been linked to the
control of the change of phase in plant development in Arabidopsis by regulating
the expression of the Squamosa promoter-binding-like (SPL) gene family (Wu and
Poethig 2006). In C. sinensis, this miRNA regulates the expression of SPL 2,4,5
and 9, allowing embryonic competence acquisition in callus cells (Wu et al. 2011).
The miRNAs 159, 164, 390 and 397 were related to globular-shaped embryo
formation while miRNAs 166, 167 and 398 were required for cotyledon-shaped
embryo morphogenesis (Wu et al. 2011).
The contrasting abundance of differential small RNAs between calli that are
competent to form embryos and those that are not is evident in sweet orange.
According to the high-throughput sequencing (HTS) analysis of small RNAs and
RNA degradome tags, 50 known and 45 novel miRNAs were identified, as well as
203 target genes (Wu et al. 2015). The abundance if miRNAs in embryogenic callus
were lower than in non-embryogenic callus, suggesting a possible repression of
important transcripts in target genes, activating the biological processes required for
differentiation.
6.4 Conclusions
Many crop plants have been sequenced; however, not all have been propagated
using SE. In the current crisis of global warming and declining arable soil, it is
urgent to secure the future of food availability. The use of SE is a powerful tool that
can be exploited in plants used for human consumption. In difference to other
methodologies, SE does not need foreign genes or expensive and sophisticated
technologies. It takes what the plant is programmed to do already and manipulates it
by using epigenetic regulation under specific conditions. The use of drugs such as
5-Aza, which has been found to promote SE and increase the number of embryos
produced, could help to solve the problems of multiplication rates in some SE
protocols. The three epigenetic mechanisms studied in many of the plants discussed
in the present chapter reveal the importance of DNA methylation, histone modifi-
cations and miRNAs to gene regulation, morphogenesis and embryo production.
Acknowledgments This work was supported by a grant received from the National Council for
Science and Technology (CONACyT 178149 to C.D.P.). F.D.A was supported by a scholarship
(255368) from CONACyT.
6 Epigenetic Advances in Somatic Embryogenesis …95

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