Regulation of Somatic Embryogenesis in Plants

REGULATION OF SOMATIC
EMBRYOGENESIS IN PLANTS
A DISSERTATION FOR THE PARTIAL FULFILLMENT OF

THE DEGREE OF MASTER OF SCIENCE IN BOTANY
UNDER THE UNIVERSITY OF KALYANI
Supervised By –
DR. BAPI GHOSH
ASSISTANT PROFESSOR
DEPARTMENT OF BOTANY (DODL)
Submitted By –
KOUSIK PAL
ROLL NO – KU/BOT4/1801273
ENROLLMENT NO – KU/MSC/BOT/0070/18
Regulation of Somatic Embryogenesis in Plants
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ACKNOWLEDGEMENT
I am extremely grateful to Dr. Bapi Ghosh, Assistant

Professor, University of Kalyani (DODL) for providing me the opportunity to
make the study in the department and utilizing the facilities during the course of
investigation.
I also express my sincere gratitude and hearty regards
to respected supervisor Dr. Bapi Ghosh, Department of Botany, University of
Kalyani (DODL) for his valuable guidance, keep interest and constant
encouragement throughout the entire period of my investigation and also
providing his lab to me throughout the investigation and necessary technical
help and suggestion during preparation of the project.
Date: 31.03.2021 Kousik Pal,

M.Sc. 4th Semester,
Cytogenetics & Plant Breeding.
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UNIVERSITY OF KALYANI
FACULTY OF SCIENCE
DEPARTMENT OF BOTANY
KALYANI 741235, WEST BENGAL
Date: 31/03/2021
To whom it may concern

This is certifying that Kousik Pal, has carried out the review work paper entitled
“Regulation of Somatic Embryogenesis in Plants” under my supervision. She has
carried out this review paper with great sincerity and labour. All the rule and
regulation laid down by the University of Kalyani for the partial fulfilment of the
Degree of Master of Science in Botany have been followed by Kousik Pal. In my
opinion this review work is worthy for the fulfilment of Master Degree of Science.
Date : 31.03.2021
Place : Kalyani, Nadia, W.B
(Dr. Bapi Ghosh)

Assistant Professor
Department of Botany
Directorate of Open and Distance Learning
University of Kalyani
Kalyani 741235, West
Bengal
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UNIVERSITY OF KALYANI
FACULTY OF SCIENCE
DEPARTMENT OF BOTANY
KALYANI 741235, WEST BENGAL
Date: 31/03/2021
This is to certify that the review work paper entitled “Regulation of Somatic
Embryogenesis in Plants” submitted by Kousik Pal is done by him/her under the
supervision of Dr. Bapi Ghosh for the partial fulfilment of the Degree of Master of
Science in Botany, University of Kalyani. Kousik Pal has presented the work
comprehensively and satisfactory and the review paper has been duly examined by
us. We recommended the review work for the partial fulfilment of the Degree of
Master of Science in Botany, University of Kalyani.
Signature of the External Examiner Signature of the Internal

Examiner
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CONTENTS
ABSTRACT------------------------------------------------------------------------ 6
INTRODUCTION---------------------------------------------------------------- 6-7
EARLY SOMATIC EMBRYOGENESIS--------------------------------------- 9-10
SOMATIC VS. ZYGOTIC EMBRYO DEVELOPMENT-------------------- 10-11
REGULATION OF SOMATIC EMBRYOGENESIS------------------------- 11-14
STAGES OF EMBRYO DEVELOPMENT------------------------------------ 14-15
FACTORS THAT INDUCED SOMATIC EMBRYOGENESIS------------- 15-17
STRESS & SOMATIC EMBRYOGENESIS----------------------------------- 17-18
SOMATIC EMBRYOGENESIS RECEPTOR KINASES--------------------- 18-20
LEAFY COTYLEDONS--------------------------------------------------------- 21
WUSCHEL------------------------------------------------------------------------ 21-22
BABY BOOM(BBM)------------------------------------------------------------- 22
OTHER FACTORS INVOLVED IN SIGNAL TRANSDUCTION
DURING THE INDUCTION OF SOMATIC EMBRYOGENESIS-------- 22-24
SOMATIC EMBRYOGENESIS INCIDENTS & VARIOUS
NETWORKS---------------------------------------------------------------------- 24-30
EPIGENETICS------------------------------------------------------------------- 31-32
APPLICATION OF SOMATIC EMBRYOGENESIS------------------------ 32
REFERENCE--------------------------------------------------------------------- 32-44
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Abstract
Somatic embryogenesis is the developmental process by which somatic cells undergo
restructuring to generate embryogenic cells. These cells then go through a series of
morphological and biochemical changes that result in the formation of a somatic or non-
zygotic embryo capable of regenerating plants. Somatic embryogenesis represents a
unique developmental pathway that includes a number of characteristic events:
dedifferentiation of cells, activation of cell division, and reprogramming of their
physiology, metabolism, and gene expression patterns. Valuable studies have elucidated
the developmental processes pertaining to the control parameters of somatic
embryogenesis. These studies have emphasized the transitional state from somatic to
embryogenic cells, identified differentially expressed genes in non-embryogenic and
embryogenic calli, isolated varieties of genes that are likely involved in the embryogenic
pathway, compared specific gene products that accumulate during different stages of
somatic embryogenesis, and discovered molecular or protein markers for somatic
embryogenesis. An understanding of embryogenic pathway initiation, and origin of
somatic embryos (unicellular or multicellular) is critical to scientific and biotechnological
applications. Cell tracking has been successfully applied to determine the fate of
embryogenic cells. Identification of hormone-inducible genes has yielded clues to the
control of gene expression during embryogenic development. Characterization of genes
taking part in signal transduction pathways, such as somatic embryogenesis receptor-
like kinases, has generated great interest in the switching of several signal cascades
during somatic embryogenesis, and has identified how genes encoding transcription
factors such as BBM, LEC1, and LEC2 could be used to regulate somatic embryo
development. Protein markers are useful probes for defining embryogenic potential and
for marking different phases in plant development. This review provides a systematic and
comprehensive analysis of current advances in the development of somatic
embryogenesis, as well as the cellular and molecular mechanism of somatic
embryogenesis in higher plants. The developmental pathway, differential gene
expression, and extracellular protein markers during somatic embryogenesis are
especially emphasized.
Introduction
Higher plant embryogenesis is divided conceptually into two distinct phases: early
morphogenetic processes that give rise to embryonic cell types, tissues, and organ
systems, and late maturation events that allow the fully developed embryo to enter a
desiccated and metabolically quiescent state. Embryogenesis is the process by which
embryo formation is initiated, either from a zygote (zygotic embryogenesis, ZE) or from
somatic cells (somatic embryogenesis, SE). ZE is carried out after the fusion of gametes.
However, the formation of asexual embryos can be induced in vitro from cells that come
from an explant of vegetal tissue. The SE process also occurs in nature. Under certain
environmental conditions such as heat and drought, the plant Kalanchoe produces,
around their leaves, small bipolar structures, which develop later in plantlets. There are
several other paths leading to the formation of an embryo. For instance, apomictic
embryogenesis takes place in the seed primordium (ovule) and the embryos produced are
genetically identical to the mother plant. Microspores can also produce embryos, and the
cells of the suspensor can change their identity to embryogenic cells when the original
embryo loses its capacity to develop.
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Somatic embryogenesis represents a complete model of totipotency and involves the

action of a complex signaling network, as well as the reprogramming of gene expression
patterns that are regulated in a specific way. This gene regulation usually is in response
to exogenous stimuli produced by the use of plant growth regulators (PGR) or certain
stress conditions, mainly low or high temperature, heavy metals, osmotic shock or
drought. The induction of SE in vitro can be accomplished through two pathways. When
SE is direct, somatic embryos are formed at the edge of an explant; when it is indirect,
SE occurs through the proliferation of a disorganized and dedifferentiated tissue called
callus.
Somatic embryogenesis has several biological and scientific advantages. For instance, it
has the potential for the improvement of plants of commercial importance, as well as for
the study of the genetic and physiological changes that are related to the fate of a plant
cell. Until now, most studies have examined the mechanisms involved in the induction of
the SE process using model plant species, such as carrot, alfalfa, corn, and rice.
However, other species, such as Arabidopsis thaliana and Gossypium hirsutum, have
been used to study the signaling pathways of the PGR action leading to the development
of plant cells.
The process of fertilization triggers the formation of an embryo and this mechanistic
process is called embryogenesis.
However, do you know fertilization is not the only process of embryo development?
Yes, it’s true! You may have heard of the phenomenon of pathogenesis, which occurs in
lower plants. Parthenogenesis is defined as the growth and development of an embryo
without fertilization of the female gamete.
Furthermore, in nature, it is observed that tissues that are part of the embryo sac, or
those that surround it, can also develop into an embryo. However, particular
environmental conditions, which exist inside the embryo, will be required for the process.
This idea led to the concept of somatic embryogenesis.
This article will pose a brief description of somatic embryogenesis, the process involved,
its application, and existing examples. So, this is a layout of the whole story and process
of somatic embryogenesis.
What is Somatic Embryogenesis?

It is defined as the development of an embryo, derived from somatic cells (cells of the
embryo sac or the cells surrounding it, other than gametes) under an appropriate
artificial environment (in-vitro). It’s a significant process of tissue culture, as it offers a
rapid large-scale propagation system that is useful for many industries.
The somatic embryos develop and differentiate the same way as the zygotic embryo. They
also pass through the proembryo, globular, torpedo (or scutellar stage in monocots), and
cotyledonary stages of development.
The other terms used for the embryo developed by this process include adventitious
embryo, accessory embryo, embryoids, embryo-like structures, etc.
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Figure: The developmental stages of the somatic embryo of carrot.
1-3: one, two, four celled stage
4-5: Globular stage
6: heart-shaped stage
7: Torpedo stage embryo
8-9: Well-formed plantlets
Source: Bajaj, Y. P. S. (1995). DOI:10.1007/978-3-662-03091-2_8
Types of Somatic Embryogenesis:

1. Primary somatic embryogenesis: In this type, the embryogenesis can only be
induced by using the explants.
2. Secondary somatic embryogenesis: In this phenomenon, the development of the
embryo is induced through existing somatic embryos.
The process of somatic embryogenesis involves four key steps which are, induction,
maintenance, development, and regeneration. (These steps will be covered in detail in a
later article.)
However, the two ways of inducing somatic embryogenesis include:
1. Direct somatic embryogenesis: In this process, the embryo is developed without

any intermediate callus stage. The embryo can be developed by directly inducing
the explant for the genesis.
2. Indirect somatic embryogenesis: In this process, the development of an embryo
occurs with an intermediate callus stage. So, it is a multistep process.
Examples of Somatic Embryogenesis

The first study of somatic embryogenesis was reported in 1958 in the carrot (Daucus
carota). Since then, the species became a model organism for the study. However, now
more than 300 studies are available for somatic embryogenesis in various plants.
Some other plants that are studied using the method are given below:
 In Ranunculus sceleratus, floral tissues are used for embryogenesis. The medium
used to induce the process is 10% coconut milk with or without IAA and it takes 3
weeks for the embryo development.
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 In loblolly pine (Pinus taeda), the cotyledonary tissue is induced for the
development of the embryo. The media, in this case, is used with 2.6 mg/L
abscisic acid, and cold storage is used to improve competence.
 To induce somatic embryogenesis in Japanese larch (Larix kaempferi), the
immature zygotic embryos are culture, the callus is collected for the formation of
the somatic embryo. The collected calli are cultured on half-strength Quoirin and
Lepoivre media containing 90 mM sucrose and 7.6 μM ABA for one month. This is
done for a higher yield.
 The somatic embryogenesis is induced in the explants of leaf and shoot apex of
Eucalyptus globulus using media containing 40 μM Picloram.
 The formation of the somatic embryo, in the East Indian sandalwood tree (S.
album L.), is induced by using nodal explant on the culture media, with 2.5 mg/L
2,4-D and 3 mg/L kinetin.
Several studies like this are present, which demonstrate the success of somatic
embryogenesis in tissue culture labs. However, as you can observe from the examples,
specific chemicals and conditions are required to induce embryo development in specific
plants.
Figure: Two different pathways of SE in dicots (i.e., direct and indirect SE), different (i.e., globular,
heart, torpedo and cotyledonary) stages of embryos,factors affecting SE are kept at bottom in oval,
and one central green oval shows some genes involved in SE. SERK1-5 (SOMATIC EMBRYO
RECEPTOR KINASE 1-5), LEC1, LEC2 (LEAFY COTYLEDON 1,2), BBM (BABY BOOM), FUS3 (FUSCA
3), ABI3(ABA INSENSITIVE 3), AGL15 (AGAMOUS LIKE 15), ASET1-3 (Alfalfa SE-specific transcripts),
AtECP31 (Arabidopsis thaliana Embryogenic31), AtECP63 (Arabidopsis thaliana Embryogenic63 cell
proteins), CaM genes (Calmodulin genes), Cdc2MS (Cell division cycle), CEM1 (elongation factor-1α),
CGS102, CGS103, CGS201 (Carrot glutamine synthetase), Dcarg1 (Daucas carrotaauxin regulated
gene), SAUR (small auxin up-regulated = Pjcw1, Top1 (topoisomerase1), DcECP31, DcECP40,
DcECP63 (Daucus carota embryogenic cell protein), H3-1, H3-11 (Histone 3), KYP/SUVH4
(Kryptonite), LBD29 (LATERAL ORGAN BOUNDARIES DOMAIN 29), PRC 1(POLYCOMB REPRESSIVE
COMPLEX1)
Source: Zimmerman Lynn J. (1993). Somatic Embryogenesis: A Model for Early

Development in Higher Plants. The Plant Cell, 5, 1411-1423.
Page No : 11
Early Somatic Embryogenesis

Once the somatic cells are induced to generate cells with embryogenic capacity, the new
cells can form structures capable of regenerating a complete plant. System suspensors
are very noticeable in gymnosperm somatic embryos. However, in many angiosperms,
suspensors are either absent or strongly reduced due to the absence of the hypophyseal
cell.
It is unclear how cells initiate embryo formation. Nonetheless, it has been established
that an irregular distribution of auxins must be established to initiate embryo formation.
This asymmetrical auxin distribution results from differential transport (Márquez-López
et al., 2018; Figure 1). In the case of ZE, an asymmetric cell division occurs, whereas in
SE this is
often not
observed. An asymmetric mitotic division of the zygote produces two different cells: one
cell gives rise to the suspensor and the other to the embryo proper. At the octant and
globular stage, protoderm formation and primordial initiation takes place. The
differential transport and asymmetrical auxin distribution continue during these stages,
giving rise to the different tissues that will form the embryo. The transportation and
accumulation of auxin produce the interaction with other factors, such as cytokinins
(CKs), which leads to the expression of specific genes.
Figure: Auxin biosynthesis during the induction of somatic embryogenesis. (A) Auxin
transport during the development of somatic embryos, globular stage (B) and heart stage
(C). Colors indicate the localization of the expression of the genes. IAA, indole-3-acetic
acid; 2, 4-D, 2, 4-Dichloroacetic acid; TAA1, TRYPTOPHAN AMINOTRANSFERASE OF
ARABIDOPSIS 1; YUC, YUCCA; PIN, PIN-FORMED; MP, monopteros.
(Source: Zimmerman, 1993; Singh, 1978; von Arnold et al., 2002)
Somatic versus zygotic embryo development
While zygotic embryogenesis (ZE) starts from a single cell followed by formation of a
globular embryo containing a determined number of cells, SE starts from a single cell or
a group of cells and attains a globular structure containing a variable number of cells.
How a group of cells initiate embryo formation is not clear, but considering our
knowledge about ZE, an asymmetric distribution of auxin must be established. Somatic
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embryo development encompasses key stages of ZE: the heart and torpedo stages in the
case of dicotyledonous species; the globular, cutellar, and coleoptilar stages in the case
of monocotyledonous species; and early and late embryogenesis in the case of
gymnosperm species. The embryo suspensor, a multicellular basally situated organ
formed during the first divisions of the zygote or at the earliest stages of SE, plays a
pivotal role in the establishment of apical–basal polarity. The suspensor is a terminally
differentiated organ subjected to gradual elimination by vacuolar PCD. Cell proliferation
and tissue patterning events in the embryo proper must be balanced by terminal cell
differentiation and death in the suspensor. The mature somatic embryos resemble
zygotic embryos morphologically and physiologically. Both exhibit apical–basal and radial
polarity, possess a primary shoot and root meristems, and contain the typical embryonic
organs radicle, hypocotyl, and cotyledons. The key genes controlling ZE perform similar
roles during SE. Somatic embryos accumulate similar nutrients required for subsequent
germination of the seedling and target them to the same cellular compartments; however,
the timing of accumulation and the exact proportion between individual types of
nutrients can vary. In addition, somatic embryos may not require desiccation (albeit that
desiccation is important for SE in Medicago sativa and Picea spp.) and may skip the
dormancy period prior to germination.
Regulation of somatic embryogenesis

Numerous studies described below (Table) demonstrate that all the key regulators of ZE
discovered in A. thaliana and other systems using a genetic approach are equally
essential for SE. While the initiation of embryogenic cultures depends on the
developmental stage of the starting plant material and factors in the growth medium
perceived by the complementary sensors in the cells, the maintenance of the
embryogenic potential during subsequent cultivation requires the simultaneous activity
of signalling and genetic pathways.
Table: Regulators of somatic embryogenesis

Name Functions Species Overexpression phenotype
Agamous-like MADS box TF A. thaliana Primary somatic

15 (AGL15) embryogenesis from
zygotic embryos and also
in the shoot apical
meristem
Baby Boom AP2/ERF family Brassica napus, A. Direct somatic
(BBM1) TF thaliana embryogenesis on
seedlings
Nicotiana tabacum Sterility if constitutive;

spontaneous
organogenesis if
inducible
Capsicum annum Indirect somatic
embryogenesis
in otherwise recalcitrant
plants
Embryomaker AP2/ERF family A. thaliana Enhanced direct and
(EMK) TF indirect
somatic embryogenesis
Cell wall components
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SE is accompanied by modifications of the structure and molecular composition of the

cell wall. These changes are thought to be important for the establishment of the balance
of forces required for the maintenance of the specific cell shape, cellular architecture,
and determination of the division plane. Apart from controlling the morphogenic events
inside the cell, the cell walls are responsible for the communication with neighbouring
cells. A plethora of signalling factors exported from the cytoplasm into the cell walls can
then spread through the apoplast to the surrounding cells and stimulate embryogenesis.
Some of these signalling factors isolated from the conditioned culture medium were not
only able to support embryogenesis in low density embryogenic cultures, which
otherwise fail to initiate embryo development, but could in addition induce embryogenic
transformation of non-embryogenic cultures.
Extracellular proteins
Several cell wall-modifying enzymes are differentially expressed during SE. For example,
induction of SE in divergent species is accompanied by up-regulation of xyloglucan
endotransglycosylases. This class of enzymes modifies the structure of xyloglucan chains
and alters the mechanical properties of the cell wall. SE of Pinus radiata is accompanied
by up-regulation of α-d-galactosidase (named SEPR1), which cleaves terminal α-
galactosyl moieties of glycolipids and glycoproteins and can thus modify the structure
and properties of the cell wall. Extracellular glycosylated acidic class IV endochitinase or
extracellular protein 3 (EP3) is required for transition from the globular to the heart stage
in carrot embryogenic cultures. The exact function of endochitinase remains unknown;
nonetheless, it appears to be a part of a phylogenetically conserved pathway, since
endochitinase from sugar beet stimulates SE in cell cultures of P. abies. Interestingly, the
endochitinase is expressed only in a subset of morphologically distinct cells in the
embryogenic cultures located outside proembryogenic masses and not in the developing
somatic embryos. In the seeds, endochitinase is not expressed in the embryos, but in the
inner integumentary cells of young fruits and in a specific subset of cells located in the
middle of the endosperm of mature seeds. These findings suggest that endochitinase is
required for the processing of signalling molecules which play a nurturing role during
both SE and ZE. The non-specific lipid transfer proteins (LTPs; Sterk et al., 1991) belong
to an abundant class of proteins found in the conditioned medium of carrot embryogenic
lines. The level of LTP expression in cotton cell lines is high before induction of
embryogenesis and during the globular stage, whereas it diminishes during post-globular
stages. The LTPs were implicated in the control of protoderm differentiation in
Arabidopsis. Correspondingly, ectopic overexpression of grapevine LTPs under the control
of the 35S promoter affects establishment of bilateral symmetry of the embryos and
disturbs epidermal cell layer morphology. At the cellular level, LTPs were implicated in
the transport of phospholipids or other non-polar molecules from the endoplasmic
reticulum to other cellular compartments in the carrot system and therefore they could
be responsible for the stabilization and transport of signalling molecules through the
apoplast and symplast. Germin-like proteins (GLPs) belong to one of the most abundant
groups of extracellular proteins found in embryogenic lines of Pinus caribaea. Several
studies showed up-regulation of transcription of GLPencoding genes in embryogenic lines
of Caribbean pine, white lupin, and wheat. In all cases their expression was limited to
embryogenic cells. GLPs belong to a cupin superfamily of ubiquitous plant proteins with
divergent primary sequences, but conserved tertiary structure. They all are hydrogen
peroxide-producing enzymes of two types: oxalate oxidase or superoxide dismutase.
Embryogenesis-related GLPs are of the oxalate oxidase type, and high oxalate oxidase
activity of GLPs was detected in the embryogenic lines of wheat. Thus, the active forms of
GLPs reside in the cell wall and can increase cell wall rigidity by producing hydrogen
peroxide, which cross-links glucuronoarabinoxylan polymers. The involvement of GLPs in
embryogenesis is further supported by the finding that expression of an A. thaliana cupin
At4g36700 is regulated by the AGL1 transcription factor, which controls both ZE and SE.
Arabinogalactan proteins (AGPs) The AGPs are a heterogeneous group of molecules
composed of a polypeptide, a long chain of branched glycan, and a lipid. Although,
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numerous studies have demonstrated a signalling role for AGPs during embryogenesis,
the molecular mechanisms behind this activity remain poorly understood. One of the
possible scenarios involves the activity of chitinases. Chitinases can cleave off the
glycosyl chains of AGPs and the released oligosaccharins can serve as signalling
molecules to promote SE in divergent systems. Chitinase EP3 co-localizes with AGPs in
developing seeds, providing further evidence that the EP3/AGPs signalling module plays
a key role in embryogenesis in carrot. In agreement with this hypothesis, inactivation of
AGPs by Yariv reagent inhibits SE. However, treatment of AGPs with the endochitinase
EP3 produces more active AGPs. This questions the role of glycosyl residues of AGPs for
the induction of SE. Furthermore, the phytocyanin-like domain of AGP from Gossypium
hirsutum (cotton) that lacks glycosylation motifs was sufficient alone to induce SE.
Judging the contribution of protein and glycan components of AGPs to the regulation of
SE is so far not possible, and more work needs to be done to reach an unambiguous
conclusion.
Figure: Comparison of somatic and zygotic embryogenesis pathways in angiosperms (A) and gymnosperms
(B). Stereotypical morphological stages of zygotic (top rows in A and B) and somatic (bottom rows in A and B)
embryogenesis. The embryo structures are drawn not to scale. In angiosperms, both zygotic (shown for
Arabidopsis) and somatic (shown for carrot; Street and Withers, 1974) embryogenesis start with asymmetric
divisions, producing cells with different fates. The apical cell proliferates, eventually giving rise to the new
plant, while the basal cell forms a terminally differentiated suspensor which disappears by the heart stage.
The zygote in gymnosperms contains free nuclei, then undergoes cellularization and divisions, and gradually
forms suspensors during the early embryogenic stage. In the somatic pathway, the polar structures
composed of proliferating cells on one pole of the embryo, and large vacuolated non-proliferating cells on the
opposite pole, appear already during proembryogeny (Filonova et al., 2000b). The vacuolated cells give rise to
the suspensor, which, similarly to the case of angiosperms, dies during maturation. Zygotic embryos in some
genera contain small rosette cells at the base of suspensor, which can divide and form aberrant embryos
(rosette polyembryony). Cleavage polyembryony, when the early embryo splits into four or more identical
embryos while only a single embryo normally develops to maturity, is a common feature of multiple genera of
gymnosperms (e.g. Pinus; Filonova et al., 2002). In SE, cleavage polyembryony was reported even in species
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which lack this feature during zygotic embryogenesis (e.g. Picea). In the latter case, the additional embryos
form from the embryonal mass cells. Both zygotic and somatic pathways are shown for P. abies (Smertenko
and Bozhkov, 2014; © Springer-Verlag Berlin Heidelberg).
Source: Leyser, O. and Day, S. (2003). Mechanisms in plant development Blackwell publishing.
somatic embryos. In the seeds, endochitinase is not expressed in the embryos, but in the
inner integumentary cells of young fruits and in a specific subset of cells located in the
middle of the endosperm of mature seeds. These findings suggest that endochitinase is
required for the processing of signalling molecules which play a nurturing role during
both SE and ZE. The non-specific lipid transfer proteins belong to an abundant class of
proteins found in the conditioned medium of carrot embryogenic lines. The level of LTP
expression in cotton cell lines is high before induction of embryogenesis and during the
globular stage, whereas it diminishes during post-globular stages. The LTPs were
implicated in the control of protoderm differentiation in Arabidopsis. Correspondingly,
ectopic overexpression of grapevine LTPs under the control of the 35S promoter affects
establishment of bilateral symmetry of the embryos and disturbs epidermal cell layer
morphology. At the cellular level, LTPs were implicated in the transport of phospholipids
or other non-polar molecules from the endoplasmic reticulum to other cellular
compartments in the carrot.
Table: Regulators of Somatic Embryogenesis
Oligosaccharins
A class of oligosaccharides called lipo-chito-oligosaccharides (LCOs) were originally
identified as nodulation (Nod) factors secreted by the bacterium Rhizobium to promote
plant cell division and formation of nodules colonized by Rhizobium. Nod factors have a
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uniform backbone made of 3–5 chitin (1,4-linked N-acetyl-dglucosamine) residues with

an N-acetyl group on the terminal non-reducing sugar substituted for an acyl chain.
Rhizobial Nod factors can stimulate SE up to the globular stage by promoting cell
division in Daucus carota and P. abies cell cultures. Embryogenic cell lines produce their
own type of LCOs with similar structure to Nod factors. The fraction of conditioned
medium enriched with these LCOs can stimulate SE.
Stages of Embryo Development

Although there is a morphological resemblance between somatic and zygotic embryos,
their development is distinctive based on plant classification (angiosperms and
gymnosperms). It is considered that zygotic embryos are nourished via the phloem
tissue, whereas somatic embryos use an exogenous supply of carbohydrates and their
morphological stages occur without vascular tissue connection.
Theoretically, plant development can be divided into two different phases: (1)
embryogenesis sensu stricto, which begins with the formation of the zygote and
concludes at the cotyledonary stage, and (2) the maturation of the seed. The somatic and
zygotic embryo developmental stages are divided into two main metabolic phases. The
first is at a morphogenetic level, where the meristem activity is triggered at a
physiological level and the process of growth, storage and maturation is initiated. The
second is a metabolic stage that is characterized by biochemical activities and the
preparation for desiccation to complete the seed formation process. In this last phase,
somatic embryos achieve both morphological and physiological maturity, which
guarantees satisfactory post-embryonic performance. Therefore, the conversion potential
is considered to be programmed during embryo maturation. However, somatic embryos
do not require desiccation.
Somatic embryo development involves similar stages to ZE, such as the globular-shaped,
heart-shaped, torpedo-shaped, and cotyledonal stages in the case of dicotyledonous
species, and globular, scutellar, and coleoptile stages in the case of monocotyledonous
species. Once the somatic embryos reach the cotyledonary stage, they initiate a shoot
meristem, and seedling growth begins.
Factors That Induce Somatic Embryogenesis

Understanding the physiological and molecular mechanisms by which the induction
(direct or indirect) of SE occurs is a crucial step for its manipulation. Several factors can
induce SE. The conditions of the culture medium, the high concentrations of PGRs, and
the wounding of explant are other types of stress that can cause plant cells to change
their cellular and molecular programs. The type of explant, the age and the genotype of
the mother plant, the physiological conditions of the incubation, and the cellular density
in the case of suspension cultures, as well as the generation of homogeneous cell
aggregates, are factors that must be considered in order to produce the acquisition of
embryogenic potential.
The source of nitrogen, as well as its concentration in the culture medium, has been
shown to be an essential element for the induction of SE. In different plant species, such
as Cucurbita pepo, Medicago sativa, Coffea arabica, and Daucus carota, it has been
determined that both nitrate and ammonium content in the culture medium have a
significant effect on the response of the explants to the induction of SE. It has been
proposed that stress is the switch that stimulates cellular reprogramming toward an
embryogenic path. However, the mechanism by which the nitrogen sources participate in
the induction of embryogenic potential remains unknown.
The Role of Plant Growth Regulators During the Induction of Somatic

Embryogenesis
Page No : 17
In plant culture systems, the addition of PGR to the culture medium plays an important
role in inducing cell differentiation, in particular during the induction of SE. Most of the
SE process depends on the concentration and kind of PGR used for each culture.
Different plant species, such as C. canephora, A. thaliana, and Musa spp. responded
successfully to the SE induction using different explants, conditions, and concentrations
of PGR.
Many species that are able to produce somatic embryos from cell suspension cultures
require the addition of auxins in the culture medium. The use of 2, 4-dichloroacetic acid
(2, 4-D) has an essential role in the induction of SE and the initial stages of development
of the somatic embryos. For example, the productivity for embryogenic date palm crops
increased 20 times by adding a low concentration of 2, 4-D. The use of auxins modified
their endogenous metabolism in a significant way; for example, in carrots, the use of 2,
4-D in the culture medium induces an embryogenic response that is associated with the
increase of the endogenous levels of indole-3-acetic acid (IAA). The pre-treatment of
plants before the induction of SE in C. canephora also modified the endogenous
metabolism of IAA.
Other PGRs, such as CKs, also participate in the development of the plants, promoting
the formation of buds, delaying the aging of the leaves and, together with the auxins,
stimulating cell division; both regulators are known to act synergistically. A high ratio
between CKs and auxins stimulates the formation of shoots while that a low ratio
induces the regeneration of roots and the proper establishment of meristems in Pisum
sativum. This two PGR can act either synergistically or antagonistically during the
induction of SE. Recent studies using synthetic reporter genes such as DR5 for auxins
and a two-component system (TCSv2) for CKs have opened a window into the molecular
mechanisms by which such interaction occurs during biosynthesis, transport and
signaling.
In recent years there has been a significant increase in the knowledge of the signal(s)
that gives rise to the SE process, but it is still unknown if auxins are the primary signal
that initiates the changes in the genetic program that leads to the production of somatic
embryos. In C. canephora, it has been shown that polar transport of the IAA is needed for
the formation of the apical-basal axis. It has also been reported that CKs are essential to
maintaining basal levels of auxin biosynthesis during root and shoot development,
suggesting that there is a homeostatic regulatory network to support adequate
concentrations between auxins and CKs in the development of the plant. It is possible
that a similar system is operating during the induction of SE. However, this must be
tested.
Plant Growth Regulator Response Genes During the Induction of

Somatic Embryogenesis
The SE process implies the integration of endogenous signals and gene reprogramming,
which unchains the signal that initiates the embryogenic process. The use of exogenous
auxins, either alone or in combination with other PGRs or stress, induces the expression
of different genes, which modify the genetic program of the somatic cells and regulate the
transition to each of the stages during the development of SE. Most of these genes belong
to one of these four categories: transcription factors (TFs), proteins that act in the cell
Page No : 18
cycle, biosynthesis of PGR, mainly auxins, as well as proteins involved in the signaling
pathway.
It is generally accepted that the SE process involves three phases: the induction of SE,
the formation of the meristematic centers, and the development of the somatic embryo.
Each stage comprises the interaction of multiple factors, e.g., external signals, changes
in the endogenous concentrations of different PGRs, and the expression of numerous
genes. Molecular studies of the induction of SE are challenging since it is difficult to
identify the cells that will become new somatic embryos. However, it is possible to carry
out bioinformatics analysis from transcriptomic studies gain a better picture of the
candidate genes involved in the initiation of the process.
Production of the signal that leads to the changes in the genetic program requires the
participation of several metabolic pathways. However, there is a consensus that auxins
play a critical role in the SE process. It is known that auxin plays a crucial role in the
formation of embryo patterns in angiosperms and in gymnosperms. During the induction
of SE in C. canephora, there is an increase in the content of endogenous IAA and in the
expression of the genes that code for the enzyme tryptophan aminotransferase
(TRYPTOPHAN AMINOTRANSFERASE OF ARABIDOPSIS 1; CcTAA1), and for the enzyme
flavin mono-oxygenase (YUCCA; CcYUC1 and CcYUC3). Both are involved in the
biosynthesis of IAA.
The response of the explant is not confined to the increase in the IAA levels. Differential
gene expression can modulate the embryogenic capacity of cells, and the number of
genes turned off in somatic cells to allow for the change from a somatic to an
embryogenic state is higher than the number of genes that are turned on. In the SE of
Arabidopsis, the modulation of several AUXIN RESPONSE FACTORS (ARF) transcripts
suggests the extensive participation of auxin signaling during the process. Almost half of
the 23 ARF genes are transcribed during SE in Arabidopsis; six of them are upregulated
and five are down-regulated. Other members of the auxin signal transduction pathway,
like the putative Aux/IAA gene from Elaeis guineensis, EgIAA9, or cotton, are also
involved in the induction of SE. An extensive analysis of gene expression during the
induction of SE in cotton shows that more than 80 genes related to the metabolism of
auxins are differentially expressed.
Stress and Somatic Embryogenesis

Somatic embryogenesis is a multifactorial event, which is the result of a series of
physiological, biochemical and molecular changes taking place in plant cells. SE requires
embryogenic competence through dedifferentiation, chromatin remodeling, programming
of gene expression, and stress events mentioned above. In general, the SE induction
includes a multitude of parallel signals that involve alterations in the levels of
endogenous PGR and stress factors.
Different studies support the theory that the first stages of SE are characterized by the
induction of numerous genes related to stress such as those discussed later on this
review. Recent evidence in potato, Pinus sylvestris, Picea asperata, Oldenlandia
umbellata, and Cyathea delgadii has revealed that the presence of different types of
stress plays an essential role in the induction of SE. The main stress for cells during the
induction of SE is the presence of high auxin concentration in the culture medium.
Page No : 19
Other stresses used for the induction of SE are extreme pH, heat-shock exposure or
treatment with various chemical substances.
Usually, the combination of physical stress with high auxin concentration in the culture
medium improves the embryogenic response. This effect was observed in Cattleya maxim
where the effect in the SE induction was evaluated using a combination of salt (0.3 M
NaCl) or osmotic stress (sorbitol 0.4 M), and the culture in a medium supplemented with
2,4-D (0.45 μM) significantly increases the percentage of protocorms with embryogenic
calli. In some angiosperms such as Panax ginseng, the treatment of somatic embryos
with abscisic acid (ABA) and polyethylene glycol (PEG) at a concentration of 20 μM and
3.75%, respectively, improve both the maturation and regeneration of somatic embryos
compared to the untreated. However, in gymnosperms, the combined application of ABA
and PEG has been shown to be necessary to stimulate the maturation and functional
development of somatic embryos. For example, in Pinus sylvestris, embryo production is
commonly induced by eliminating auxin from the culture medium, ABA addition and
subsequently a PEG drying step. In P. strobus, variable amounts of water at the
beginning and during the cultivation phase influences the maturation response of the
embryos. Meanwhile, changes in water availability either by solutes or physical
restriction can affect the maturation response in some conifers. Other types of stress like
heat-shock induce the SE in Gladiolus hybridus. In cotton, several of the genes expressed
during the induction of SE are related to the homeostasis of auxins and ethylene, as well
as several related-stress TFs.
Transcription Factors and Signal Transduction Involved in

Somatic Embryogenesis
There is very little current information on whether the genes involved in the induction of
SE work independently or in a network-like structure. However, the analysis of the
interaction among different clusters of genes shows that they can act in parallel or in
sequence. The use of transcriptomics has provided valuable. Indicates that the genes
expressed during the induction of SE are divided into the categories of stress-related
genes, PGR-related genes, and TFs.
The changes in the genetic program of the cells that lead to the induction of SE require
the regulation of several genes. In both angiosperms and gymnosperms, little is known
about gene expression, the early stages of embryogenesis, which is crucial for the later
development of the embryo. For example, it has been reported that in conifers such as
Araucaria angustifolia that the expression patterns of AaSERK1 during SE are very
similar to SERK1 homologs of angiosperms. These changes require the substantial
participation of TFs. Plant genomes contain a large number (6–10%) of TFs-coding genes.
Some of these TFs are shared among a variety of plant species. Among the TFs that have
been found during the induction of SE in different species are ABAINSENSITIVE 3 (ABI3),
AGAMOUS LIKE (AGL), BABY BOOM (BBM), CUP SHAPED COTYLEDONS (CUC), FUSCA3
(FUS3), LEAFY COTYLEDON (LEC), LEAFY COTYLEDON LIKE (LIL), SOMATIC
EMBRYOGENESIS RECEPTOR-LIKE KINASE1 (SERK1), RWP-RK DOMAIN-CONTAINING 4
(RKD4)/GROUNDED (GRD). VIVIPAROUS1 (VP1), and WUSCHEL (WUS). In conifers,
several homologs of important genes that participate during ES have been found, such as
SERK1, LEC1, and WOX2, but it is still unknown whether they present patterns and
expression functions similar to angiosperms. Several of these genes are also expressed
Page No : 20
during the formation of zygotic embryos. The application of auxins or their analogs, like
2, 4-D, enhances the expression of several TFs, such as BBM, WUS, and VP1 during the
induction of SE.
In some cases, like the SE induced in wounded tissues, there is a signal that occurs
before to the expression of the TFs listed in the last paragraph. The expression of
WOUND INDUCED DEDIFFERENTIATION1 (WIND1) TF, from the AP2/ERF family, is
required before the expression of LEAFY COTYLEDON2 (LEC2) takes place. The
expression of some TFs is specific to particular species; however, several others are
expressed in all the systems of induction of SE studied. The roles of these TFs in the
signaling process are discussed below.
Somatic Embryogenesis Receptor Kinases (SERK)

Among the different genes that increase their expression during the induction of SE,
SERK is the most relevant. This family of TFs is involved in a range of developmental
processes that include differentiation/trans differentiation and cellular totipotency.
The first SERK gene was identified in D. carota. It was detected in embryogenic cultures
in the early days of culture in the presence of 2, 4-D. This gene is expressed in cells that
develop in somatic embryos until the globular stage, just before the transition from the
differentiation state to the development state. The expression of SERK increases several
times in the embryogenic cells of A. thaliana, Citrus unshiu, Dactylis glomerata, G.
hirsutum, Helianthus annuus, Medicago truncatula, Solanum tuberosum, Vitis vinifera,
Cocos nucifera, Oryza sativa, Theobroma cacao, Triticum aestivum, Zea mays,
Cyrtochilum loxense, and A. angustifolia.
The evidence of the participation of SERK in the induction of SE has emerged from the
analysis of gene expression. For example, SERK1 is highly expressed during the
formation of embryogenic cells in in vitro culture of A. thaliana and in all of the cells of
the developing embryo during early SE, up until the heart stage of the somatic embryo.
After this stage, the expression of SERK1 is no longer detectable in the embryo. However,
in seedlings that over-expressed SERK1, the mRNA exhibited a 300–400% increase in the
efficiency of the initiation of SE. These results suggest that an increase in the expression
levels of SERK1 confers embryogenic competence to cells in culture. In O. sativa, SERK2
is expressed almost three times more in the embryogenic callus and maturation stage
than in the non-embryogenic callus. These results suggest that different members of the
SERK family have unique functions. Similar results have been found in T. aestivum. In
this plant, members of the SERK family are expressed differentially in response to
different PGR sensitivities; i.e., SERK2 and SERK3 elicit auxin-specific responses while
SERK1 and SERK5 may be mediated by the signaling pathway of brassinosteroids.
In addition to auxins, other factors modified the expression of SERK. In M. truncatula, the
expression of SERK1 is stimulated by the presence of auxin, but not by CKs. However,
when the CKs are co-administered with auxin, the level of expression of SERK1 increases
synergistically compared to the up-regulation of auxin alone. In response to a higher
level of expression of SERK, the number of embryogenic calluses increase as well as the
formation of somatic embryos.
Table: SOMATIC EMBRYOGENESIS RECEPTOR KINASE (SERK) gene regulating

embryogenesis in different studied plant materials
Page No : 21
Name of the plant Common Name SERK Gene

Adiantum capillus-veneris Maidenhair fern AcvSERK
Ananas comosus Pineapple Ac SERK1–3
Arabidopsis thaliana Thale cress At SERK1–5
Citrus sinensis Orange Cs SERK
Citrus unshiu Tangerine Cu SERK
Cocos nucifera Coconut Cn SERK
Cucurma alismatifolia Summer tulip CaSERK
Cyclamen persicum Persian cyclamen Cp SERK1–2
Cyrtochilum loxense Not available Cl SERK
Dactylis glomerata Orchard grass DgSERK
Daucus carota Carrot Dc SERK
Dimocarpus longan Longan Dl SERK
Garcinia mangostana Magnosteen Mangosteen SERK
Glycine max Soya bean Gm SERK1–2
Gossypium hirsutum Cotton Gh SERK1–3
Helianthus annuus Sunflower HaSERK
Marchantia polymorpha Common liverwort Mp SERK
Medicago truncatula Barrel clover Mt SERK1–6
Musa acuminata Banana MaSERK
Nicotiana benthamiana Tobacco Nb SERK3A, Nb SERK3B
Ocotea catharinensis Not available OcoteaSERK
Oryza sativa Rice OsbiSERK, Os SERK, Os
SERKlike1, Os SERKlike2
Physcomitrella patens Moss Pp SERK1–3
Poa pratensis Common meadow grass Poap SERKlike1–2
Populus trichocarpa Black cottonwood Pp SERK1-4
Prunus persica Peach Persica SERK
Prunus salicina Japenese plum PsSERK
Rosa canina Dog rose RcSERK
Rosa hybrid Hybrid tea rose RhSERK1–4
Selaginella moellendorffii Club moss Sm SERK1–4
Solanum lycopersicum Tomato Sl SERK1, Sl SERK3A, Sl
SERK3B
Solanum peruvianum Wild tomato Sp SERK
Solanum tuberosum Potato St SERK
Sorghum bicolor Sorghum Sb SERK1–3
Theobroma cacao Cocoa tree TcSERK
Triticum aestivum Wheat Ta SERK1, Ta SERK2, Ta
SERKlike3
Vitis vinifera Grape Vv SERK1–3
Zea mays Maize Zm SERK1–3
Leafy Cotyledon (LEC)
Another important participant in the regulation of SE and plant embryo development is

the LEC family of TFs. LEC1 has an essential role in ZE and has been suggested to
control diverse processes in seed development, including embryo morphogenesis,
maturation phases, germination, and early and late embryogenesis; it also appears to
allow the formation of the embryo by establishing an embryonic environment. LEC1 is
also involved in photosynthesis and chloroplast biogenesis early in seed development,
and seed maturation late in the development of zygotic embryos. This gene network
Page No : 22
regulated by LEC1 has been conserved in dicotyledonous plants that diverged tens of
millions of years ago.
LEC1 and LEC2 were the first TFs shown to induce SE when ectopically expressed in
seedlings. The auxin-dependent upregulation of LEC2 has been associated with the
induction of SE, whereas LEC2 expression was markedly lower in non-embryogenic
callus of A. thaliana, suggesting that LEC2 mediates the increase in the endogenous
auxins observed during the induction of SE. Similar results were found in T. cacao,
where LEC2 is highly expressed in the embryogenic callus and its overexpression in
cotyledon explants increased the embryogenic response. The ectopic overexpression of
LEC2 from Ricinus communis in A. thaliana induces the expression of TFs such as LEC1,
L1L, FUS3, ABI3, and WRINKELED1 (WRI1). Also, the expression of the fatty acid
elongase 1 (FAE1) and, in consequence, an accumulation of triacylglycerols, especially
those containing the seed-specific fatty acid, eicosenoic acid (20:1 Δ11), in vegetative
tissues was observed.
WUSCHEL (WUS)
The establishment of the shoot apical meristem (SAM) is essential for SE and for shoot
regeneration. These processes require the expression of WUS, which encodes a
bifunctional homeodomain TF. WUS contains a highly conserved homeobox domain, and
at the conserved C terminal region it has three functional domains: an acidic domain, a
WUS-box (TLPLFPMH), and an EAR-like motif. A very important characteristic of WUS is
its ability to move from one tissue to another. It can move from its biosynthesis site, the
central zone (CZ), into the daughter cells in the peripheral zone, where it activates the
transcription of CLAVATA3 (CVL3), a negative regulator. CLV3 moves into the
extracellular space and binds to CLV1, which in turn inhibits the transcription of WUS.
This WUS-CLV feedback system establishment maintains the stem cell pool and the
development of SAM. Therefore, WUS has been proposed to be essential for SE and in
vitro shoot regeneration.
WUSCHEL, like LEC2, responds to the presence of auxins. Auxins trigger a signaling
cascade that initiates the vegetative-to-embryogenic transition, and this transition is
mediated by WUS. The gradient of auxins that is detected during the pre-treatment of C.
canephora plantlets and later during the initial phases of SE correlates with the induced
WUS expression during SE in A. thaliana.
It has been observed that WUS-related genes are up-regulated during SE in different
species, such as Ocotea catharinensis, M. truncatula, G. hirsutum, and C. canephora. In
C. canephora, overexpression of WUS enhances SE in heterologous systems, increasing
the somatic embryo production by 400%. In G. hirsutum, the ectopic expression of
AtWUS promotes the proliferation and differentiation of transgenic callus and positively
regulates LEC1, LEC2, and FUS3. WUS overexpression enhances the induction of SE and
can improve regeneration in cotton, and its overexpression in A. thaliana roots, leaf
petioles, stems, or leaves induces the formation of somatic embryos.
Baby Boom (BBM)
Another key regulator of plant cell totipotency is BBM. BBM can induce embryogenesis in
differentiated cells and could be a vital factor in plant embryogenesis development. BBM
Page No : 23
triggers a set of genes like LEC1 and LEC2, as well as ABI3 and the FUS3 network, which
together activate SE. The induction of SE by BBM is a dose-dependent mechanism and
regulates the transcription of significant embryo identity genes.
The BBM family encodes APETALA 2/ETHYLENE RESPONSE FACTOR (AP2/ERF) DNA-
binding type TFs identified in the gymnosperms, angiosperms, algae, and mosses, these
TFs act as a network regulation in response to biotic and abiotic stress. The AP2/ERF
domain can bind to a GCC box, a DNA sequence involved in the ethylene response.
AP2/ERF are divided according to the number of AP2 domains that they contain, which
are classified into subfamilies as the Dehydration-responsive 427 element-binding
(DREB), ERF, AP2, and RELATED TO ABI3/VP1 (RAV) genes. Because RAV genes include
another DNA-binding domain, B3, RAV genes are sometimes treated as a third group in
the AP2/ERF family. The distinct feature of the BBM and BBM-like proteins is the
presence of a conserved bbm-1 motif (GLSMIKTW) that is absent in other proteins of the
euANT lineage. BBM activated the expression of a broad set of genes encoding proteins
with potential roles in transcription, cellular signaling, cell wall biosynthesis and
targeted protein turnover, such as the ACTIN DEPOLYMERIZING FACTOR9 (ADF9).
In A. thaliana and B. napus, BBM changes its spatial-temporal expression in the early
stages of embryogenesis. Some reports show that BBM is expressed in the heart state of
an embryo and root development and enhances the proliferation of somatic embryos.
This response is also produced by ectopic expression of BBM, which changes from
vegetative to embryonic growth and induces spontaneous SE in these two species. The
heterologous expression of BBM from A. thaliana and B. napus in Nicotiana tabacum
produced an increase in the regeneration capability. In Capsicum annum, both LEC1 and
BBM are expressed and show high levels of expression in the different phases of
development of the somatic embryo.
On the other hand, it is worth highlighting that BBM can show differential expression
depending on the species and the embryogenic protocol. In a study using two species of
the genus Coffea, it was found that while in C. arabica a BBM-like gene showed a twofold
change in expression in embryogenic cell suspension in comparison to embryogenic calli,
in C. canephora BBM1 expression was only observed after SE induction. It has been
found that the BBM gene is expressed at higher levels during SE in comparison to ZE in
T. cacao, and its overexpression in A. thaliana and T. cacao led to phenotypes associated
with SE that did not require exogenous hormones. However, BBM overexpression can
inhibit the subsequent development of the somatic embryos in T. cacao, while the BBM
overexpression in Populus tomentosa induced SE.
Other Factors Involved in Signal Transduction During the

Induction of Somatic Embryogenesis
Somatic embryogenesis signaling is a very complex process where several molecular

players are involved; it would be tedious to list them all. However, there are two other
major factors that need to be mentioned. One is the intervention of 14-3-3 proteins,
which participate in several processes such as the development of the seeds and during
the induction of SE in Carica papaya. The other factor actively involved during the SE
induction, process, and development is epigenetic.
Page No : 24
14-3-3 Adaptor Proteins

14-3-3 adaptor proteins are a group of proteins involved in the signal transduction
pathway that is shared by several PGRs involved in SE induction. These proteins are
highly conserved phosphoserine-/phosphothreonine-binding proteins, discovered in the
brain of mammals in 1967, with a subunit mass of 30 kDa.
In plants the number of members of these proteins is variable (Figure 2). There are 13
14-3-3 adaptor proteins in Arabidopsis, six in cotton, 17 in tobacco, ten in tomato, five in
barley, and eight in rice.
Figure: Phylogenetic tree for 14-3-3 genes family in several species. The sequences of
Coffea canephora GF14 were obtained from http://coffee-genome.org. Rice sequences
were obtained in http://rice.plantbiology.msu.edu. Tomato sequences were obtained in
https://solgenomics.net/. Arabidopsis sequences were obtained in
https://www.arabidopsis.org/. The sequences were aligned in the software MEGA 7
http://www.megasoftware.net/. The percentage of replicate trees in which the associated
taxa clustered together in the bootstrap test (1000 replicates) is shown next to the
branches. The analysis was conducted in MEGA7 using the Neighbor-Joining method.
Abbreviations: Os, Oryza sativa; Sl, Solanum lycopersicum; Cc, Coffea canephora; At,
Arabidopsis thaliana.
The use of proteomics techniques has illuminated the changes in hundreds of proteins,
including the family 14-3-3, during the induction of SE. Some 14-3-3 proteins are
abundant in the embryogenic tissues of Cyclamen persicum, and Larix principis. In oak,
these proteins are more abundant in proliferating embryos than in mature embryos.
An excellent example that shows the role of 14-3-3 proteins in the induction of SE is
protein phosphatase 2A (PP2A). This enzyme consists of a catalytic subunit and a
regulatory A subunit together with a third variable B subunit. The B subunit is the
Page No : 25
component that determines the substrate specificity and subcellular localization of

PP2As. PP2A is a complex enzyme. In A. thaliana, there are 25 genes involved in the
transcription of PP2A three subunits. The catalytic subunit (PP2Ac) is coded by five
genes, three other genes encoding A subunits and seventeen different genes encoding B
subunits. The subunit A is essential for auxin transport, while the 65 kDa regulatory
subunit of PP2A has regulatory functions. The subunit A has been associated with the
SE process. There is a noticeable increase in phosphorylation of specific proteins in
embryogenic cultures compared to the non-embryogenic cells of C. persicum, which has
been correlated with higher levels of PP2A and a 14-3-3-like protein. Other components
of the signal transduction cascade, such as G proteins and calreticulin, increased during
cyclamen SE. It has been suggested that the increase in the regulatory subunit of PP2A
and 14-3-3 proteins during the induction of SE is related to the stress conditions
produced by the in vitro culturing of C. persicum and L. principis embryogenic cultures.
Somatic embryogenesis incidences and various networks
Embryogenesis and woody genera

In certain plant groups like woody genera, response is poor in developing callus and
embryogenic tissues; the exudation of phenolics and similar other compounds aggravate
the problem further. With the growing knowledge and other technological advances,
these problems were overcome in many plants, and consequently, many woody plants
are now cultured in vitro. But most of the woody plants are still either completely
reluctant or respond poorly to treatments for embryogenesis. With the current high
demand for woody plants (due to medicinal, aesthetic values, food, fiber, timber, fuel),
plant conservation concerns and climate change attract researchers’ attention in
unveiling new strategies for rapid, mass propagation of such plants. Marker-assisted
breeding, genetic transformation, etc. are also being targeted to improve plant quality. SE
is one of the methods being continuously upgraded and renovated to suit plant
propagation particularly for those plants that have a long-life cycle, produce less/no
seeds, and do not reproduce vegetatively. This technique is preferred over the
organogenesis because of bipolar embryo that does not need separate treatment for root
or shoot induction. The bipolar embryonal axis has both shoot and root ends and is
directly grown to complete plants. Various factors govern SE induction and embryo
numbers such as plant genotype, type of explants, type and strength of stimulus, and
age of tissue (e.g., juvenility). After acquisition of embryogenic competence, embryo
development may not always reach the final stages of plantlet formation. In plants, where
embryos developed, a similar developmental pattern was observed for the attainment of
other developmental stages. Thus, SE is suitable for forest and other groups of plant
propagation, genetic engineering, and cryopreservation of elite germplasm.
Genes regulating vegetative to embryonic (early stage)

Transition initial steps of direct SE which is not true for indirect SE in BABYBOOM
(BBM)-mediated LAFL [LEC1/LEC1-LIKE (L1L), ABSCISIC ACID INSENSITIVE 3 (ABI3),
FUSCA3 (FUS3), and (LEC2)] gene expression. Chromatin state of LAFL gene is one of the
factors that determine direct or indirect SE. LEC1/LEC1-LIKE (L1L) and LEC2 induce
direct SE when constitutively overexpressed, while LEC1 in particular is detected later
after embryo appears on the callus surface.
Role of plant growth regulators (PGRs) in embryogenesis network

PGRs play a key role in both zygotic and somatic embryogenesis. Among all PGRs, auxin
is most effective in the induction of SE. Once SE is induced, auxin concentration is either
to be lowered or completely omitted. Different PGRs, their concentrations and
combinations have different effects on the process of SE depending on the plant species.
Page No : 26
In most species, auxin, cytokinin, abscisic acid (ABA), and jasmonic acid (JA) are the key
factors triggering the embryogenic response as these have a regulatory effect on cell
cycle, division, and differentiation. Auxin 2,4-dichlorophenoxyacetic acid (2,4-D), either
alone or in combination with cytokinins, is used to induce somatic embryo in many plant
species using seeds or zygotic embryos as explants. Synthesis of jasmonic acid and
abscisic acid (stress-related PGRs) was reported in Medicago sativa throughout the
process of SE but differentially biosynthesized in different phases of SE. Gibberellins
(GAs), usually gibberellic acid (GA3), have a repressive role on the induction of SE in
some plants as it significantly upregulates gibberellins 2-oxidase (GA2ox6), repressing
GA synthesis (Elhiti et al. 2010). LEAFY COTYLEDON 1 (LEC1) is a key player in abscisic
acid (ABA)-mediated expression of YUCCA10 (YUC10) in seedlings. YUC mutants (YUC
genes are involved in auxin biosynthesis) are less responsive to secondary SE, suggesting
that the endogenous auxin is important for this process. Adventitious shoot formations
induced in short auxin exposure while somatic embryo formation in long auxin exposure.
This suggests the developmental continuum in somatic embryo and adventitious shoot
formation, where critical threshold auxin signalling is crucial in in vitro induction and
maintenance of embryo identity. Auxin-mediated plant development involves changes in
expression of auxin responsive genes, encoding a family of transcription factors, AUXIN
RESPONSE FACTORs (ARFs). The ARFs regulate the expression of target genes by
binding to AUXIN RESPONSE ELEMENT (AuxRE) TCTCTC motif, present in promoters of
auxin-responsive genes. The ARFs bind promoters via a B3-type DNA binding domain,
specific to plants. Molecular studies of Arabidopsis thaliana identified about 22 ARF
genes and a pseudogene. Among the different ARFs, ARF5, ARF6, ARF7, ARF8, and
ARF19 activate the target gene expression, while ARF1, ARF2, ARF3, ARF4, and ARF9
repress the expression of target genes. Wójcikowska and Gaj observed upregulation of
four ARFs (ARF5, ARF6, ARF10, and ARF16) during the inductive phase of SE in
Arabidopsis, while two ARFs (ARF8 and ARF17) were upregulated in advanced stages. A
number of ARFs are being identified in different plants, and intensive research continues
in this field to elucidate their role in plant developmental processes.
Plant genotype, explants, and oxygenation determining embryogenesis

The success in regenerating plant via SE is largely dependent on the genotype of the
plant species. Different plant parts respond differently, while cultured in vitro or even
different genotypes of a plant behave uniquely/differently. Sané et al. reported that
Ahmar and Amsekhsi cultivars were more callogenic than Tijib and Amaside, exhibiting
response differences in primary callogenesis in different date palm cultivars. Similarly,
woody plants are more recalcitrant in showing responses than the herbaceous groups of
plants. Various types of explants are used for generating somatic embryos in different
plants. The type and size of explant and plant species significantly influence the process
of SE. Kocak and co-workers demonstrated that the leaves and petioles of Cyclamen
persicum were more responsive compared to the ovule and ovary and took less time to
induce callus; in carnation, callus followed by somatic embryos were obtained from petal
explants in a number of cultivated varieties.The dissolved oxygen concentration in
culture flask has significant influence on the development of somatic embryos. It is
observed that the concentration of oxygen in suspension had ostensible effects on the
maturation process and the number of embryos. The 50% dissolved oxygen (DO) levels in
medium showed maturated embryos with lower numbers, while at 80% DO
concentration, opposite response (i.e., higher embryo numbers with less maturity) were
noted in Coffea arabica.
Somaclonal variation, SE, and genetic integrity

Somaclonal variation (SV) is a phenomenon whereby the variations are manifested
among the tissue culture raised plants, and these variations include both phenotypic
and genotypic alterations. The genetic alterations occur spontaneously under stressed
microenvironment and can continue to remain for several generations. The changes are
Page No : 27
heritable and non-LAFL network genes [LEAFY COTYLEDON1, LEC1/LEC1-LIKE (L1L),

ABSCISIC ACID INSENSITIVE 3 (ABI3), FUSCA3 (FUS3), and (LEC2)] are involved in the
heritable containing point mutation, chromosomal deletion, substitution, DNA breakage,
and ploidy. The PGR-induced stress, nutrient, osmotic, humidity transpiration
imbalances, oxidative stress, and light stress are the forces generating these
abnormalities. Non-heritable genetic changes constitute some of the epigenetic changes,
which are less stable, remain for a lesser period of time, and disappear on the cessation
of stress condition. DNA methylation, hypo- and hyperacetylation led some of the
epigenetic changes occurring in in vitro-cultivated plant cells. Polycomb protein group
modifies histone, and these proteins form conserve regulatory complexes that modify the
chromatin state and gene expression during cellular transition from somatic to
embryogenic cells. Two of
such conserved regulatory complexes are Polycomb repressive complex 1 (PRC1) and
PRC2. Trimethylation of histone 3 (H3K27me3) lysine 27 through SET-domain protein
and subsequent binding of PRC1, which carry out ubiquitination of 119 lysine residues
of histone H2A, improves compactness of the chromatin. The state of chromatin
determines binding of regulatory protein complexes and influences expression of genes.
In SV, the frequency of variations increases with the age of cultures, number of
subcultures, and duration of stress. The variations noted in plants regenerated through
SE have both advantages and disadvantages. SV is a big problem where plants’ genetic
and phenotypic integrity and purity are aimed at. In such cases, the genetic purity is
ensured by taking the explants from authenticated, registered sources while the SV is
also widely used in plant improvement programs. The easily available variations among
the regenerated plants could be profitable only when maintained stably for generations.
The main problem of SV is the non-beneficial, redundant, and unstable variations,
restricting the progress of breeding, and most of the regenerated plants showed poor
agronomic performance.
Carbohydrates and underlying mechanism of SE

The reprogramming of signalling and communication of callus cells seem to be chemical
in nature, and the analysis of callus exudates in the medium shows compounds like
sugars, growth regulators, low molecular weight compounds, amino acids, and vitamins.
Different carbohydrates were used as energy source in various media, of which sucrose
and glucose are observed to be the most efficient for better cultural growth. In some
plants, SE is absent until sucrose was added to the media, confirming its importance in
embryo induction. For example, the expanded cotyledons of melon were noted to induce
somatic embryos only in the presence of sucrose. Sucrose or glucose may be substituted
by other carbohydrates as carbon sources depending upon the tissue, plant, and species
from which explants are taken. Grzyb et al. noted many fold effects of increased soluble
sucrose at developmental transition to SE expression phase. Species specific storage
products are also accumulated during SE process and are absent in other stages of
development.
Somatic Embryogenesis Receptor Kinase, SERK, and other genes regulating SE
SERK is involved in embryogenic competence acquisition; the gene encodes protein and
was isolated initially from carrot, named as DcSERK. Later, SERK homologues were also
reported in many other plants (Table).
Table: Genes/orthologs regulating somatic embryogenesis in various investigated plants

Genes/orthologs Encoded products and Investigated plant
possible role
ABI3 (ABA INSENSITIVE 3) B2, B3 domain Arabidopsis thaliana
transcription factors;
regulate embryo-specific
ABA-inducible genes
Page No : 28
AGL15 (AGAMOUS LIKE MADS-box transcription Arabidopsis thaliana

15) factor; promote somatic
embryogenesis
ASET1-3 (Alfalfa SE- Specific transcript, Medicago sativa
specific transcripts) (product unknown);
expressed at early stages of
embryogenesis
AtECP31, AtECP63 Embryogenic 31 and 63 A. thaliana
cell proteins; expression
during torpedo stage of
embryogenesis, ABA-
responsive genes
BBM (BABY BOOM) AP2/ERF Transcription B. napus
factors; activates LEC1-
ABI3-FUS3-LEC2 network
to induce somatic
embryogenesis
CaM (Calmodulin genes) Kinase type protein; Many plants
accumulates during early
embryogenesis through Ca-
mediated signalling
Cdc2 (Cell division cycle 2) Cdc protein; regulation of M. sativa
cell cycle progression
CEM1 Polypeptide, similar to Daucus carota
translational elongation-
factor 1α; Expressed
strongly pro-globular and
globular stage
CGS102, CGS103, CGS201 Glutamine synthetase; D. carota
enzyme, expression during
early SE stages
DcARG1 (Auxin regulated Protein specific to auxin; D. carota
Gene 1) expression at early
induction stage
DcECP31, DcECP40, Embryogenic cell protein; D. carota
DcECP63 expression at torpedo stage
of SE
FUS3 (FUSCA 3) Transcriptional factor A. thaliana
family protein; regulate
synthesis of storage
proteins and lipids
H3-1, H3-11 (Histone 3,11) H3-1 gene transcript, auxin M. sativa
responsive
Kryptonite (KYP/SUVH4) Methyl transferase; role in A. thaliana
dedifferentiation
LATERAL ORGAN Transcription factor; A. thaliana
BOUNDARIES DOMAIN 29 dedifferentiation of cells,
(LBD29) role in early embryogenesis
LEC1, LEC2 (LEAFY B3 domain transcription A. thaliana
COTYLEDON 1,2) factor; essential for somatic
embryogenesis
PICKLE ATP-dependent chromatin A. thaliana
remodeler; inhibits SE
PJCW1, PJCW2 = SAUR, Protein product, influence Glycine max
SMALL AUXIN UP- cell elongation
Page No : 29
REGULATED GENE
POLYCOMB REPRESSIVE Epigenetic effector A. thaliana
COMPLEX1 proteins; stem cell self-
(PRC 1) renewal, pluripotency, gene
silencing; repressive effect
on dedifferentiation
ability of cells
PRIMORDIA TIMING Gene product; help in flower A. thaliana
development; increases SAM
cell population
SERK1-5 (SOMATIC EMBRYO Receptor like kinase protein; Many plants
RECEPTOR KINASE 1-5) acquisition of embryogenic
competence
TOP 1 (Topoisomerase1) Constitutively expressed D. carota
during cellular proliferative
activities and at torpedo stage
of SE development
WUSCHEL Homeo-domain transcription A. thaliana
factor; Promote “vegetative to
embryonic” transition
Structurally, SERK consists of serine - proline-rich leucine zipper, kinase domain, signal
peptide, leucine-rich region, transmembrane domain, and C-terminal region. SERK, a
cell surface receptor, triggers a signal cascade after binding to the ligand through the
leucine-rich repeat (LRR) domain and with the help of intracellular domains reaches to
the nucleus. This cascade alters gene expression pattern via chromatin remodelling.
Activity of genes is often altered either by repressing specific or selective genes and
activating/changing the expression of others. SERK overexpression is observed during
embryogenic induction till the globular stage and together with other genes like BBM and
LEC promotes transition to embryogenic cells from non-embryogenic tissues. LEAFY
COTYLEDON (LEC) is one among the most important genes, playing a central role in
both zygotic and somatic embryogenesis. Loss of functional mutation in LEC largely
impaired the embryonic development. The LEC mutant shows significantly reduced or
total repression of embryogenic response as observed in double and triple mutants in A.
thaliana. The impairment is most ostensible in the maintenance of embryonic cell fate
and specification of cotyledon identity. Overexpression of LEC2 affects several target
genes including the AGAMOUS-like 15 (AGL15) TF gene and auxin pathway genes. LEC2
mutants do not acquire desiccation tolerance and do not accumulate storage reserves in
cotyledon tips. Studies suggested that FUSCA3 (FUS3), LEC1, and LEC2 do not play a
major role in the induction of SE, but during late stages of embryogenesis, their function
has a significant say. Watery callus and root hairs are produced in LEC1 single mutant,
while LEC1 and FUS3 double and triple mutants negatively affect the SE process.
Embryo identity and maturation are regulated by the network of LAFL proteins
LEC1/LEC1-LIKE (L1L), ABSCISIC ACID INSENSITIVE 3 (ABI3), FUSCA3 (FUS3), and
(LEC2) where B9 and B3 domains are encoded by LEC1 and LEC2 genes, respectively.
B9 is a subunit of NUCLEAR factor Y (NF-Y-B9), and B3 is a domain which contains
transcription factor LEC2 playing a role in maintaining the morphology of suspensor,
progression via maturation phase, cotyledon identity specification, and suppressing
premature germination. Accumulation of storage macromolecules, desiccation tolerance,
and cotyledon development are defective in zygotic embryos where loss of function
mutation occurs in LAFL genes. LAFL proteins regulate the expression of BBM which
gets reduced in case of LAFL mutant seeds. LEC2 have central role in maturation phase
of SE; LEC2 up regulates AGL15 which is involved in the formation of somatic embryos
from embryogenic tissues like zygotic embryos. AGL15 and LEC2 are involved in the
activation of INDOLE-3-ACETIC ACID INDUCIBLE 30 (IAA30) which when mutated
affects the AGL15-mediated SE that normally shows enhancement under its effect.
Embryo development is switched on in the vegetative cells that acquire embryogenic
Page No : 30
competence under the influence of ectopic expression of LEC. The LEC genes in turn
seem to be regulated by PICKLE by causing chromatin remodelling, repressing the
embryonic identity regulators during germination. BABYBOOM (BBM) is a transcription
factor of AINT EGUMENTA-LIKE (AIL) APETALA2/ethylene-responsive element
(AP2/ERF) family, isolated from Brassica napus embryos developed from pollen grains.
Ectopic expression of BBM in A. thaliana seedlings induces somatic embryos without the
exogenous stress or growth regulator treatment. BBM along with other AP2/ERF family
of transcription factors help in maintaining meristematic state of shoot and root
meristems. It regulates cell growth and identity and promotes morphogenesis and
cellular proliferation by exploiting AIL and LAFL proteins while mediating embryogenesis.
Ectopic expression of BBM has an inductive effect in the formation of “somatic embryo-
like structures” in Arabidopsis. BBM in SE binds to YUCCA3 (YUC3), YUC8, and
TRYPTOPHAN AMINOTRANSFERASE OF ARABIDOPSIS1 (TAA1) and promotes auxin
biosynthesis, suggesting its role in endogenous auxin synthesis. FUS3 and LEC1
mutants completely abolish BBM-induced SE, suggesting their crucial role in BBM-
induced SE pathway. Beside adventitious root, shoot formation, and SE induction,
neoplastic growth (cell proliferation), deformed flowers, and leaves are the pleiotropic
phenotypes of BBM. In Theobroma cacao, a higher level of TcBBM expression was noted
during somatic embryogenesis than during zygotic embryogenesis time. BBM also
transcriptionally regulates LEC, FUSCA3 (FUS3), and ABI 45 INSENSITIVE3 (ABI3) genes
and induces cellular totipotency through LAFL network during seed germination. BBM
regulates the expression of AGL15 and LAFL by binding to promoter of genes. This is
evident from the observation where AGL15 and LEC2 mutants show reduced BBM-
mediated SE. Other genes like LATE EMBRYO ABUNDANT (LEA) are noted to be
abundantly expressed during later phases of embryogenesis. The LEA proteins are
hydrophilic and are regulated by ABA. The LEA proteins influence the developmental
processes of zygotic and somatic embryogenesis and also to stress-related responses. In
almost all instances, their expression is observed in embryogenic tissue and not in
vegetative cells. In addition to LEA proteins, some other genes like WUSCHEL are active
during SE; WUS develops somatic embryos indirectly, and ectopic expression of WUS
also produces somatic embryo directly and promotes organogenesis on exogenous auxin-
amended or PGR-free cultures as evidenced in WUS mutants. The emergence of shoots
forming embryos similarly occurs in ectopically expressed WUS explants in auxin-free
and CLAVATA (CLV) mutants in 2,4-D (auxin)–added medium. WUS and CLV normally
function to maintain stem cells and cell differentiation in shoot meristem. Cell
differentiation is also regulated by these genes in the shoot apical meristem (SAM) of CLV
mutants where somatic embryos are formed by some non-committed cells. WOUND
INDUCED DEDIFFERENTIATION1 (WIND1) or RAP2-4 (Protein RELATED to APETALA2
4) induces SE and play a role in callus formation in tissue damage and wounding.
PLETHORA2 (PLT2) plays a major role in the induction and specification of root pole in
SE. Reverse glycosylating protein (RGP-1), a membrane protein, encourages plant cell
wall development by facilitating polysaccharide metabolism, and in early phases of
somatic embryogenesis, it is thought to participate in structural reorganization.
AGAMOUS-like 15 (AGL 15) is isolated as a MADS-box gene, detected in many plants
(e.g., B. napus, Arabidopsis, Taraxacum), and in alfalfa, it is detected in somatic
embryos. AGL15 regulates the expression of several genes during the process of SE by
encoding MADS-box family of transcription factors. For example, AtGA2ox6 is encoded
by a gene, controlled by AGL15. Overexpression of AGL15 induces SE in embryogenic
tissue like zygotic embryos and could not induce SE spontaneously in Arabidopsis
seedlings. Ectopic expression of AGL15 under CaMV35S promoter induces embryo
formation in seedling in which 2,4-D and AGL15 both regulate expression. Among the
different RKD (RWP-RK domain containing proteins), only RWP-RK DOMAINCONTAINING
4 (RKD4) is noted to produce embryos; RWP-RK DOMAIN-CONTAINING 4 (RKD4)/
GROUNDED (GRD) also induces embryos and is thought to be expressed in maximum in
suspensors and early stages of embryos. On the overexpression of RKD4, SE develops
into seedlings by stimulating root cells to proliferate; and in RKD4 mutants, embryo
Page No : 31
development is arrested, and suspensor remains short. Different genes/transcription

factors (TFs) playing various roles at different stages of embryogenesis are shown in Fig.
2.
Figure: Different genes at different stages of SE pathway. Triangle 1 in yellow shows genes involved in
dedifferentiation; triangle 2 shows genes involved in acquisition of totipotency by the cells; and triangle 3
shows genes expressed in commitment of totipotent cells to embryogenic state. AUXIN RESPONSE FACTOR
19 (ARF19), POLYCOMB REPRESSIVE COMPLEX 1 (PRC1), REVERSIBILY GLYCOSYLATED POLYPEPTIDE 1
(RGP1), HEAT SHOCK PROTEIN 17 (HSP17), SOMATIC EMBRYOGENESIS LIKE RECEPTOR KINASE
(SERK1), LEAFY COTYLEDON1 (LEC1), GALACTOSIDASE BETA 1 (GLB1), WUSCHEL (WUS), CURLY LEAF
(CLF), CYCLIN DEPENDENT KINASE A1 (CDKA1), PROPORZ1 (PRZ1), SHOOT MERISTEMLESS (STM)
The mystery behind the SE is being gradually unfolded by the use of molecular
approach. Over 700 TFs and genes are being extensively studied during the process of
SE in Arabidopsis thaliana and other plants, suggesting the very significant role of TF in
competence acquisition via embryogenic reprogramming. Some of the genes and TFs
having a role in SE. Studies suggest that the basic mechanism behind the somatic and
zygotic embryogenesis is the same, and the genes regulating zygotic embryogenesis have
very similar effect on SE. Differentially expressed genes DEG1 and DEG2 associated with
embryogenesis were identified in Dactylis glomerata; DEGs express in the embryogenic
leaf (not in non-embryogenic cells) and is noted in both directly and indirectly induced
cultures, while DEG2 expression is noted only in directly induced tissues. The ectopic
expression of various zygotic embryogenic genes significantly increased the somatic
embryo development in several investigated plants. Similarly, the chromatin remodelling
determines spatial and temporal expression of genes and influences the development of
SE to a large extent. Indirect SE requires more extensive chromatin modification than
that of direct SE as was shown by differential expression of chromatin modifiers after
2,4-D–mediated callus formation.
Epigenetics
In recent years, epigenetic mechanisms during chromatin remodeling have emerged as

critical factors in SE. Epigenetic modifications are an essential part of the signaling
Page No : 32
pathway that leads to changes in the genetic program of the cells and the development of
somatic embryos. There is evidence that shows that changes in the chromatin are able to
control totipotency in plant cells. The level to which chromatin reprogramming is
required before SE induction depends on several factors, such as origin of the explant,
the culture medium, the genetic background of the mother plant, and especially the
amount of PGR used.
DNA methylation is important for somatic embryo development. In general, higher global
DNA methylation has been found in non-embryogenic cultures of Pinus radiata, P. nigra,
Rosa x hybrid, and Eleutherococcus senticosus, while low global DNA methylation has
been found in embryogenic cultures of several plants. In Quercus alba DNA is
demethylation during the induction of SE, as well as during the generation of pro-
embryogenic mass, but it gradually increases as the embryo is developing. Similar
results were observed during the SE of C. canephora, where the proembryogenic mass
had lower DNA methylation, while the maturation of the embryos was marked by a
gradual increase in the global levels of methylation.
In A. thaliana it was found that both de novo DNA methylation and maintenance of it are
required for the regulation of SE, and similar results were found in Picea abies. Changes
in the global DNA methylation pattern during long-term subcultures could lead to the
loss of the embryonic potential of proembryogenic masses.
In order to prove that in fact DNA methylation is strongly related to SE, pharmacological
experiments have been conducted in several plant species. The application of 5-
azacitidine (5-AzaC; a demethylating agent) decreased the levels of global DNA
methylation in A. thaliana and inhibited the induction of SE. Similar results have been
found in M. truncatula, D. carota, and C. canephora. Furthermore, LEC1, LEC2, and BBM
genes were up-regulated in the drm1drm2 and drm1drm2cmt3 mutants, an upregulation
that was related to an improvement in the SE response. In T. cacao, DNA methylation
increased during the induction of SE, and treatment with 5-AzaC led to the recovery of
SE potential in aged cultures. 5-AzaC is not the only drug used to disrupt epigenetic
modifications; trichostatin A (TSA), the function of which is inhibiting histone
deacetylases (HDACs), has a positive effect on gene expression. The inhibition of HDACs
has also led to an increase in the number of haploid embryos produced by heat stress in
B. napus. In fact, the treatment with TSA of germinating spruce somatic embryos
preserves their embryogenic nature. In the double mutant hda6/hda19, the upregulation
of LEC1, FUS3, and ABI3 genes was evident in germinating Arabidopsis seeds. These
double mutants also led to the production of somatic embryos in the leaves of
Arabidopsis.
Histones’ posttranslational modifications have been implicated in the formation of

somatic embryos. Histone deacetylation may also play a role in the reprogramming of
cells in the early stages of SE, since the levels of histone acetylation and the activity of
HDACs change in response to the presence of exogenous PGR during the induction of
SE.
There are several tissue-specific events involving H3K27me3. The loss of this mark
upregulates the auxin pathway and its increase leads to the repression of leaf identity.
Polycomb repressive complex 2 (PRC2) is involved in the methylation of lysine 27 in
histone H3. Double mutants of the PRC2 gene, which functions as a histone
Page No : 33
methyltransferase, CLF and SWN or VERNALIZATION 2 (VRN2) and EMBRYONIC

FLOWER2 (EMF2) form callus on the shoot apex, lead to indirect somatic embryo
formation and ectopic roots. A PRC2 mutant root hairs fail to maintain their
differentiated state and form unorganized cell masses and eventually somatic embryos
from callus. The effect of silencing genes of the PCR2 family in inducing SE depends on
the explant. In tissues where PCR2 is scarcely active, the production of somatic embryos
is efficient; however, in the tissues where it is highly expressed somatic embryos do not
form.
Application of somatic embryogenesis

The process of somatic embryogenesis serves several purposes and involves multiple
applications that are covered in this section.
1. It is a preferred in-vitro propagation method for woody plants. Here, it plays a

critical role in clonal propagation, synthetic seed production, germplasm
conservation, and cryopreservation.
2. It is used for rapid large-scale propagation of plants for the production of
secondary compounds and drugs.
3. It is an ideal system for the basic studies of plant cell biology and embryo
development.
4. It also provides a better system for the understanding of the differentiation and
mechanism of totipotency expression in plant cells.
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Regulation of Somatic Embryogenesis in Plants

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REGULATION OF SOMATIC

A DISSERTATION FOR THE PARTIAL FULFILLMENT OF

I am extremely grateful to Dr. Bapi Ghosh, Assistant

Date: 31.03.2021 Kousik Pal,

To whom it may concern

(Dr. Bapi Ghosh)

Signature of the External Examiner Signature of the Internal

Somatic embryogenesis represents a complete model of totipotency and involves the

What is Somatic Embryogenesis?

Figure: The developmental stages of the somatic embryo of carrot.

1-3: one, two, four celled stage

4-5: Globular stage

7: Torpedo stage embryo

8-9: Well-formed plantlets

Source: Bajaj, Y. P. S. (1995). DOI:10.1007/978-3-662-03091-2_8

Types of Somatic Embryogenesis:

However, the two ways of inducing somatic embryogenesis include:

1. Direct somatic embryogenesis: In this process, the embryo is developed without

Examples of Somatic Embryogenesis

Source: Zimmerman Lynn J. (1993). Somatic Embryogenesis: A Model for Early

Early Somatic Embryogenesis

(Source: Zimmerman, 1993; Singh, 1978; von Arnold et al., 2002)

Somatic versus zygotic embryo development

Regulation of somatic embryogenesis

Table: Regulators of somatic embryogenesis

Agamous-like MADS box TF A. thaliana Primary somatic

Nicotiana tabacum Sterility if constitutive;

Cell wall components

SE is accompanied by modifications of the structure and molecular composition of the

Table: Regulators of Somatic Embryogenesis

uniform backbone made of 3–5 chitin (1,4-linked N-acetyl-dglucosamine) residues with

Stages of Embryo Development

Factors That Induce Somatic Embryogenesis

The Role of Plant Growth Regulators During the Induction of Somatic

Plant Growth Regulator Response Genes During the Induction of

Stress and Somatic Embryogenesis

Transcription Factors and Signal Transduction Involved in

Somatic Embryogenesis Receptor Kinases (SERK)

Table: SOMATIC EMBRYOGENESIS RECEPTOR KINASE (SERK) gene regulating

Name of the plant Common Name SERK Gene

Leafy Cotyledon (LEC)

Another important participant in the regulation of SE and plant embryo development is

Baby Boom (BBM)

Other Factors Involved in Signal Transduction During the

Somatic embryogenesis signaling is a very complex process where several molecular

14-3-3 Adaptor Proteins

component that determines the substrate specificity and subcellular localization of

Somatic embryogenesis incidences and various networks

Embryogenesis and woody genera

Genes regulating vegetative to embryonic (early stage)

Role of plant growth regulators (PGRs) in embryogenesis network

Plant genotype, explants, and oxygenation determining embryogenesis

Somaclonal variation, SE, and genetic integrity

heritable and non-LAFL network genes [LEAFY COTYLEDON1, LEC1/LEC1-LIKE (L1L),

Carbohydrates and underlying mechanism of SE

Table: Genes/orthologs regulating somatic embryogenesis in various investigated plants

AGL15 (AGAMOUS LIKE MADS-box transcription Arabidopsis thaliana

development is arrested, and suspensor remains short. Different genes/transcription

In recent years, epigenetic mechanisms during chromatin remodeling have emerged as

Histones’ posttranslational modifications have been implicated in the formation of

methyltransferase, CLF and SWN or VERNALIZATION 2 (VRN2) and EMBRYONIC

Application of somatic embryogenesis

1. It is a preferred in-vitro propagation method for woody plants. Here, it plays a