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Víctor M. Loyola-Vargas
Neftalí Ochoa-Alejo Editors
Somatic
Embryogenesis:
Fundamental
Aspects and
Applications
Somatic Embryogenesis: Fundamental Aspects
and Applications
Neftalí Ochoa-Alejo
Editors
Somatic Embryogenesis:
Fundamental Aspects
and Applications
123
Editors
Víctor M. Loyola-Vargas Neftalí Ochoa-Alejo
Unidad de Bioquímica y Biología Molecular Departamento de Ingeniería Genética
de Plantas de Plantas
Centro de Investigación Científica Centro de Investigación y de Estudios
de Yucatán Avanzados del Instituto Politécnico
Mérida, Yucatán Nacional
Mexico Irapuato
Mexico
ISBN 978-3-319-33704-3 ISBN 978-3-319-33705-0 (eBook)

DOI 10.1007/978-3-319-33705-0
Library of Congress Control Number: 2016937943
© Springer International Publishing Switzerland 2016

This work is subject to copyright. All rights are reserved by the Publisher, whether the whole or part
of the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations,
recitation, broadcasting, reproduction on microfilms or in any other physical way, and transmission
or information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar
methodology now known or hereafter developed.
The use of general descriptive names, registered names, trademarks, service marks, etc. in this
publication does not imply, even in the absence of a specific statement, that such names are exempt from
the relevant protective laws and regulations and therefore free for general use.
The publisher, the authors and the editors are safe to assume that the advice and information in this
book are believed to be true and accurate at the date of publication. Neither the publisher nor the
authors or the editors give a warranty, express or implied, with respect to the material contained herein or
for any errors or omissions that may have been made.
Printed on acid-free paper
This Springer imprint is published by Springer Nature

The registered company is Springer International Publishing AG Switzerland
Contents
1 Somatic Embryogenesis. An Overview . . . . . . . . . . . . . . . . . . . . . 1

Víctor M. Loyola-Vargas and Neftalí Ochoa-Alejo
Part I Basic Aspects

2 The History of Somatic Embryogenesis . . . . . . . . . . . . . . . . . . . . . 11
3 The Many Ways of Somatic Embryo Initiation . . . . . . . . . . . . . . . 23
Attila Fehér, Dóra Bernula and Katalin Gémes
4 Transcriptome Profile of Somatic Embryogenesis . . . . . . . . . . . . . 39
José E. Cetz-Chel and Víctor M. Loyola-Vargas
5 Transcription Factors in the Regulation of Somatic
Embryogenesis. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53
Katarzyna Nowak and Małgorzata D. Gaj
6 Epigenetic Advances in Somatic Embryogenesis
in Sequenced Genome Crops . . . . . . . . . . . . . . . . . . . . . . . . . . . . 81
Fátima Duarte-Aké and Clelia De-la-Peña
7 The Current Status of Proteomic Studies in Somatic
Embryogenesis. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 103
Martín Mata Rosas, Francisco Quiroz-Figueroa, Laura M. Shannon
and Eliel Ruiz-May
8 Plant Fidelity in Somatic Embryogenesis-Regenerated Plants . . . . . 121
Etienne Hervé, Guyot Romain, Beulé Thierry,
Breitler Jean-Christophe and Jaligot Estelle
v
vi Contents
9 The Relationship Between Stress and Somatic Embryogenesis . . . . 151

Geovanny I. Nic-Can, Johny R. Avilez-Montalvo,
Randy N. Aviles-Montalvo, Ruth E. Márquez-López,
Erika Mellado-Mojica, Rosa M. Galaz-Ávalos
and Víctor M. Loyola-Vargas
10 The Role of the Auxins During Somatic Embryogenesis. . . . . . . . . 171
Geovanny I. Nic-Can and Víctor M. Loyola-Vargas
Part II Somatic Embryogenesis of Basic Models and Industrial

and Agronomical Crops
11 Somatic Embryogenesis in Arabidopsis . . . . . . . . . . . . . . . . . . . . . 185
Barbara Wójcikowska and Małgorzata D. Gaj
12 Maize Somatic Embryogenesis: Agronomic Features
for Improving Crop Productivity . . . . . . . . . . . . . . . . . . . . . . . . . 201
Verónica Garrocho-Villegas, Erika Almeraya
and Estela Sánchez de Jiménez
13 Somatic Embryogenesis in Annatto (Bixa orellana L.) . . . . . . . . . . 213
Elyabe Monteiro de Matos, Andréa Dias Koehler,
Daniele Vidal Faria, Ludmila Nayara de Freitas Correia,
Viviane Santos Moreira, Ana Claudia Ferreira da Cruz,
Tatiana Souza Moraes, Diego Ismael Rocha,
Virgínia Lúcia Fontes Soares, Vespasiano Borges de Paiva Neto,
Marcio Gilberto Cardoso Costa and Wagner Campos Otoni
14 Somatic Embryogenesis in Capsicum spp. . . . . . . . . . . . . . . . . . . . 233
15 Somatic Embryogenesis in Coffea spp. . . . . . . . . . . . . . . . . . . . . . 241
Víctor M. Loyola-Vargas, Johny R. Avilez-Montalvo,
Randy N. Avilés-Montalvo, Ruth E. Márquez-López,
Rosa M. Galaz-Ávalos and Erika Mellado-Mojica
16 Somatic Embryogenesis in Agave spp. . . . . . . . . . . . . . . . . . . . . . . 267
Benjamín Rodríguez-Garay
17 Somatic Embryogenesis in Agave: An Overview . . . . . . . . . . . . . . 283
Kelly Maribel Monja-Mio and Manuel L. Robert
18 Somatic Embryogenesis in Cocos nucifera L. . . . . . . . . . . . . . . . . . 297
Luis Sáenz-Carbonell, Mayra Montero-Cortés, Teresa Pérez-Nuñez,
Alfonso Azpeitia-Morales, Antonio Andrade-Torres,
Iván Córdova-Lara, José Luis Chan-Rodríguez,
Gabriela Sandoval-Cancino, Gustavo Rivera-Solis
and Carlos Oropeza-Salín
Contents vii
19 Somatic Embryogenesis for More Effective Breeding and

Deployment of Improved Varieties in Pinus spp.: Bottlenecks
and Recent Advances . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 319
Marie-Anne Lelu-Walter, Krystyna Klimaszewska, Célia Miguel,
Tuija Aronen, Cathy Hargreaves, Caroline Teyssier
and Jean-François Trontin
20 Direct and Indirect Somatic Embryogenesis in Mango
Ginger (Curcuma amada Roxb.) . . . . . . . . . . . . . . . . . . . . . . . . . . 367
Appakan Shajahan, Chellappan Soundar Raju,
Chandrasekaran Thilip, Kandhan Varutharaju,
Kunnampalli Faizal, Valiyaparambath Musfir Mehaboob
and Abubakker Aslam
21 Somatic Embryogenesis in Banana, Musa ssp. . . . . . . . . . . . . . . . . 381
Rosa María Escobedo-GraciaMedrano,
Adrián José Enríquez-Valencia, Muhammad Youssef,
Pablo López-Gómez, Carlos Iván Cruz-Cárdenas
and José Roberto Ku-Cauich
22 Somatic Embryogenesis in Jatropha curcas . . . . . . . . . . . . . . . . . . 401
Rupesh Kumar Singh, Eliel Ruiz-May, Luz María García-Pérez
and Francisco R. Quiroz-Figueroa
Part III Applications and Techniques

23 The Uses of Somatic Embryogenesis for Genetic
Transformation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 415
24 Somatic Embryogenesis in Temporary Immersion Bioreactors . . . . 435
Kelly Maribel Monja-Mio, Miguel Ángel Herrera-Alamillo
and Manuel L. Robert
25 Application of Somatic Embryogenesis to Secondary
Metabolite-Producing Plants. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 455
Felipe A. Vázquez-Flota, Miriam Monforte-González
and María de Lourdes Miranda-Ham
26 Histology and Histochemistry of Somatic Embryogenesis . . . . . . . . 471
Diego Ismael Rocha, Ewa Kurczyńska, Izabela Potocka,
Douglas André Steinmacher and Wagner Campos Otoni
27 Laser-Assisted Microdissection to Study Global
Transcriptional Changes During Plant Embryogenesis. . . . . . . . . . 495
Ricardo A. Chávez Montes, Joanna Serwatowska
and Stefan de Folter
Chapter 1
Somatic Embryogenesis. An Overview
Víctor M. Loyola-Vargas and Neftalí Ochoa-Alejo
Abstract Somatic embryogenesis is one of the most powerful tools in plant

biotechnology. It can be used to produce plants commercially, or to carry out basic
studies of cell differentiation, gene expression, molecular genetics, and many oth-
ers. We present here a compilation of the different chapters of this book.
1.1 Introduction
Initially, the necessity of solving important fundamental questions in plant biology,

such as the cell theory and totipotency, led to the development of plant cell, tissue,
and organ culture (PTC). However, nowadays PTC represents a set of very pow-
erful biotechnological tools. The applications of PTC include commercial micro-
propagation of agronomically important plant species, production of haploid and
double-haploid plants and disease-free plants, rescue of hybrid embryos or somatic
cell fusion from intra- or inter-generic sources for the production of novel hybrid
plants, induction of genetic or epigenetic variation for the production of variant
plants, and more recently the genetic engineering of plants to produce new varieties,
resistant to pests and diseases, as well to improve the quality and quantity of a
particular product obtained from a plant. Other applications include genetic mod-
ification to produce plants that can remove toxic compounds or test its toxicity
(bioremediation) (Hannink et al. 2001; Krämer and Chardonnens 2001), and the use
V.M. Loyola-Vargas (&)

Unidad de Bioquímica y Biología Molecular de Plantas,
Centro de Investigación Científica de Yucatán, Calle 43 no. 130,
Col. Chuburná de Hidalgo, CP 97200 Mérida, Yucatán, Mexico
e-mail: vmloyola@cicy.mx
N. Ochoa-Alejo
Departamento de Ingeniería Genética, and Departamento de Biotecnología y Bioquímica,
Centro de Investigación y de Estudios Avanzados del Instituto Politécnico Nacional,
Unidad Irapuato, Km 9.6 libramiento norte carretera Irapuato-León, 36821 Irapuato,
Guanajuato, Mexico
e-mail: nochoa@ira.cinvestav.mx
© Springer International Publishing Switzerland 2016 1

V.M. Loyola-Vargas and N. Ochoa-Alejo (eds.), Somatic Embryogenesis:
Fundamental Aspects and Applications, DOI 10.1007/978-3-319-33705-0_1
2 V.M. Loyola-Vargas and N. Ochoa-Alejo
of micropropagated shoots to maintain plant viruses. Root cultures can be used to

study the interaction of roots with nematodes or mycorrhizas. Recently, plants have
been modified by genetic engineers with the objective to increase the yield of
cellulose or oil for the production of biofuels (Gressel 2008; Stokstad 2012;
Takahashi and Takamizo 2012). Somatic embryogenesis is at the core of some of
these biotechnological applications and is the focus of this book.
Gottlieb Haberlandt in 1902 (Haberlandt 1902) set the theoretical basis for plant
tissue culture. He proposed that a single cell should eventually be capable of giving
origin to a complete and functional plant. This theory has been proved to be right.
At the core of this proof is the somatic embryogenesis.
1.2 Somatic Embryogenesis
Plant somatic embryogenesis (SE) is a biological process with both amazing basic
and applied aspects. SE occurs in nature. At the edges of the leaves of several
species of Kalanchoë appear small bipolar structures, some of them produce these
structures constitutively, others by the action of environmental stress, and a third
group is a combination of both (Garces and Sinha 2009).
In vitro plant cells can undergo dedifferentiation or redifferentiation to enter a
new biological program that gives rise to somatic embryos. This process has raised
one of the most important biological questions: which signals change the genetic
program of a somatic cell and make it an embryogenic cell?
Numerous factors that affect SE have been investigated in order to understand
the basis of this process and manipulate it to develop and establish efficient plant
regeneration protocols as a fundamental step for its biotechnological application
(Loyola-Vargas et al. 2008). A differentiated and specialized somatic plant cell or a
group of somatic cells with specific functions must receive a stimulus from a set of
plant growth regulators (PGRs), mainly auxins, perceive it, and then initiate the
transduction to the nucleus where the specific regulatory and structural genes will
be transcribed and subsequently will be translated into proteins involved in the
differentiation that ultimately will lead to the formation of a new somatic embryo.
All these changes can be followed at morphological, ultrastructural, genetic,
physiological, biochemical, and molecular levels.
The idea of this book is to look somatic embryogenesis in an integrative way
covering from the historical aspects of somatic embryogenesis to its applications.
It is important to know about the history of those researchers whose contributions
led to the development of this field. In Chap. 2 we describe the main facts that led
to the historical first papers on SE (Miettinen and Waris 1958; Reinert 1959;
Steward et al. 1958).
There are several pathways to initiate somatic embryogenesis (Chap. 3). Unlike
the initial belief that all plant cells are totipotents, it has been seen that it is
necessary to create appropriate conditions for some of them to regain totipotency.
Among the several factors that play a role in the induction of SE are the plant
1 Somatic Embryogenesis. An Overview 3
growth regulators, mainly auxins (Altamura et al. 2016). It is interesting that many
species require an initial shot of auxins, but thereafter the auxin must be degraded
for SE to proceed (Chap. 10) (Altamura et al. 2016). Clearly the onset of SE
depends on a complex network of interactions among plant growth regulators,
mainly auxins and cytokinins, during the early proembryogenic stages. Ethylene
and gibberellic and abscisic acids pass to play a major role during the late stages of
development. Together, the PGRs regulate multiple genes temporally and spatially
which release the changes in the genetic program of somatic cells, as well as
regulating the transition between each embryonic developmental stage.
In addition to phenotype, the origin of the explant, and genetic background of
the plant, several stress treatments such as heavy metals, low or high temperature,
osmotic shock, among others, might play a crucial role in SE induction, even in the
absence of exogenous PGRs (Chap. 9) (Cabrera-Ponce et al. 2015; Ochatt and
Revilla 2016; Salo et al. 2016).
An important concern is the fidelity of the somatic embryogenesis-regenerated
plants (Chap. 8). There is an epigenetic reprogramming during the SE and the
presence of somaclonal variation among the regenerated plants (De-la-Peña et al.
2015; Mahdavi-Darvari et al. 2015; Nic-Can et al. 2015; Solís et al. 2015). This
variation can be the result of chromosomal aberrations, genetic alterations, epige-
netic regulations, and transposable elements. The variation can be exploited for
good, as selecting stress-tolerant somaclones (Bobadilla Landey et al. 2013;
Us-Camas et al. 2014).
Beyond the biotechnological application of SE, it can be used to study the very
different aspects of its induction and the development of somatic embryos. An
aspect that is central to the study of SE is histology. SE has become an appropriate
method for studying the morphophysiological and molecular aspects of cell dif-
ferentiation (Chap. 26). The understanding of the developmental events during the
induction phase as well as the development of somatic embryos is essential to
regulate and improve each stage of the SE program efficiently. Anatomical and
ultrastructural studies may be useful for the development of protocols more efficient
for SE induction, as well as for the cellular mechanisms involved in the acquisition
of competence for SE (Konieczny et al. 2012; Quiroz-Figueroa et al. 2002).
The molecular aspects of SE have been studied extensively. In this book, several
authors have revised the most recent advances in the field. Transcriptomics of
several species has been carried out during the induction of SE and the development
of the somatic embryos (Chap. 4). Cotton is the species most studied, but the
number of species investigated by this technique is growing every day. The pattern
that is emerging from these studies suggests a predominant role of auxins during the
induction of SE, as well as for genes like LEC, WUS, FUS, and a set of transcription
factors (Shi et al. 2016; Tao et al. 2016; Trontin et al. 2016). The Next Generation
Sequencing platforms of nucleic acids can be used together with techniques that
allow the isolation of a specific cellular type, such as laser-assisted microdissection.
Together these two techniques give us a closer approach to the state of the cell in
determined space and time (Chap. 27).
The extreme changes required for the transcription of the genome during the
change of a somatic cell to an embryogenic cell need a very active participation of
transcription factors. In Chap. 5, authors made an extensive analysis of the state of
the art in relation to the participation of transcription factors in this process. An
interesting finding is that the most frequent transcription factors found active during
the induction of SE belong to the pathways of the metabolism of growth regulators,
stress, and flower development (El Ouakfaoui et al. 2010; Guan et al. 2009).
Among all the different mechanisms that regulate the expression of the genes,
epigenetics also plays an important role (Chap. 6). Different reports suggest that
auxins, in conjunction with the in vitro conditions modify the DNA methylation
status in the embryogenic cells. These changes in DNA methylation patterns are
associated with the regulation of several genes involved in SE, such as WUS,
BBM1, LEC, and several others (De-la-Peña et al. 2015).
After the genes are expressed, all the weight of the process is on the proteins.
Posttranslational modifications, protein turnover, and protein–protein interactions
are common processes associated with the regulation of proteins. All of them are
present during the induction of SE and development of somatic embryos. Proteomic
studies carried out while the SE has begun to show the deep mechanism that works
during the induction of SE (Chap. 7). One key question is if there is a common
protein pattern among different species during the induction of SE (Campos et al.
2016; Mukul-López et al. 2012; Tchorbadjieva 2016).
SE has been induced in many different species; many of them crops of commercial
interest. In the second part of the book, the SE of several important crops is analyzed:
Agave spp., Bixa orellana, Capsicum spp., Coffea spp., Curcuma, Musa spp., Zea
mays, lipid-producing plants like Cocus nucifera and Jathropha curcas, conifers such
as Pinus spp., and model plants as Arabidopsis thaliana (Chaps. 11–22).
The two major applications of SE are scale-up propagation (Chap. 24) and genetic
engineering (Chap. 23). Among the different systems to scale up the process of SE,
the temporary immersion system has some advantages. It can be automatized to
reduce labor and costs, and at the same time to produce high-quality plantlets (Fei and
Weathers 2014, 2016; Ibaraki and Kurata 2001). The SE process is a very efficient
method to regenerate transgenic plants. SE has been used in conjunction with
Agrobacterium spp., particle bombardment, and chemical-mediated genetic trans-
formation protocols. All the major annual and perennial crops, as well as model
plants, have been transformed using efficient SE systems (Arroyo-Herrera et al. 2008;
Bouchabké-Coussa et al. 2013; Canché-Moor et al. 2006; Palomo-Ríos et al. 2012).
Another application analyzed is the use of SE to produce secondary metabolites.
The production of secondary metabolites by plants requires highly specialized
tissues and a fine regulation and coordination in time and development by the plant
(De Luca and St Pierre 2000). In nature, several plant species synthesize and store
secondary metabolites in the zygotic seed, suggesting that somatic embryos can do
it. In Chap. 25 the most recent discoveries related to the accumulation of secondary
metabolites by somatic embryos are presented (Aslam et al. 2010, 2011; Sharma
et al. 2015).
1.3 Concluding Remarks
PTC in general and SE in particular have turned into an invaluable tool to plant
scientists. PTC has been commercialized around the world, and different companies
are using plant tissue culture techniques for the massive propagation of plants. The
use of PTC and the development of the omics and epigenetics have allowed the
understanding of the basic biological process.
The use of SE leads to the understanding of differentiation, as well as to the genetic
mechanisms involved in the transition from one stage to the next. Also it has led to the
isolation of genes, proteins, and metabolites involved in the cell differentiation pro-
cess. The combination of SE and genetic engineering will accelerate the discovery,
isolation, and characterization of genes involved in different cellular processes.
From the agronomy side, the most important challenges ahead are the generation
of resistant plants to pathogens and pests, as well as to abiotic stresses, increments
Fig. 1.1 Somatic embryogenesis process in different species. a Somatic embryogenesis in a leaf
of Coffea canephora. b Embryogenic mass of Cocus nucifera. c Different developmental stages of
Musa acuminata x Musa balbisiana genome AAB, subgroup Plantain. d Embryogenic mass of
Agave tequilana. Picture a is from the laboratory of Víctor M. Loyola-Vargas. Pictures b–d are
gifts from the laboratories of Carlos Oropeza-Salín, Rosa M. GraciaMedrano from Centro de
Investigación Científica de Yucatán and Benjamín Rodríguez-Garay from Centro de Investigación
y Asistencia en Tecnología y Diseño del Estado de Jalisco, respectively
in yields in commercial crops, the production of better raw material for biofuel
production, as well as the generation of plants capable of absorbing toxic com-
pounds from the environment. In all these cases, SE will play an important role
(Fig. 1.1).
Acknowledgments The work of the laboratory of VMLV was supported by a grant received from
the National Council for Science and Technology (CONACyT 157014).
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Part I
Basic Aspects
Chapter 2
The History of Somatic Embryogenesis
Abstract Somatic embryogenesis is used currently as a powerful tool in biotech-

nology. It is also used to study the development of the embryo. Somatic embryo-
genesis is a natural phenomenon that was moved from nature to the laboratory
by man. The history of the study of somatic embryogenesis is plenty of discoveries
of very different natures: from the role of growth regulators, mainly auxins, to the
function of the components of the media of culture. In this chapter, a revision of
the major contribution to the advance of knowledge of somatic embryogenesis
is made.
2.1 Introduction
Embryos, essentials for the reproduction of higher plants, were an important

improvement introduced by land plants, even before the development of seeds. This
sequence of events probably leads to the production of embryos from different
kinds of cells (Radoeva and Weijers 2014). (1) Zygotic embryos originate from
gametic cells. (2) Somatic cells give origin to somatic embryos. (3) Cells from the
seed primordium can develop into an embryo and subsequently in a seed without
fertilization in a process known as apomixis. (4) There is also embryo formation
from microspores; in the process called androgenesis, the microspores can form
haploid or doubled embryos after the use of different kinds of stresses. (5) In the
earliest steps of zygotic embryogenesis, there is a connection between the proper
embryo and the maternal tissue, this assembly is known as suspensor. This structure
is usually formed by a few cells; however, in some cases suspensor can be gen-
erated by thousands of cells and be able to form an embryo (Yeung and Meinke
1993).
V.M. Loyola-Vargas (&)

Unidad de Bioquímica y Biología Molecular de Plantas, Centro de Investigación
Científica de Yucatán, Calle 43 no. 130, Col. Chuburná de Hidalgo, CP 97200 Mérida,
Yucatán, Mexico
e-mail: vmloyola@cicy.mx

12 V.M. Loyola-Vargas
From these different ways to produce embryos, somatic embryogenesis (SE) has
become a powerful tool to study and understand the mechanisms of the development
of the embryo. In combination with traditional agricultural techniques, SE is used for
the propagation of vegetable species of commercial interest and their genetic
improvement (Loyola-Vargas et al. 2008). The onset of SE requires a set of inter-
actions between auxins, ethylene, cytokinins, and several other growth regulators to
change the genetic program of the cells (Jiménez 2005). Also, other factors can
influence the induction and development of somatic embryos, such as the origin of
the explant, the physiology of the mother plant, the environmental conditions of the
incubation, and the composition of the culture medium (Loyola-Vargas et al. 2008).
A scientific discovery is the result of different efforts, many times from diverse
lines of research or thinking. Since the landmark paper by Haberlandt (1902) pro-
posed the theory of totipotency, several research groups were working on the
development of plant tissue culture. Almost 60 years later, the discovery of SE was a
consequence of the level of development of plant tissue culture at the end of the 50s
of the last century. Calli and suspension cultures were well established, and the study
of cytokinins (Miller et al. 1955) and auxins (Skoog 1947) was under development.
Before the classical papers by Waris (Krikorian and Simola 1999; Miettinen and
Waris 1958), Steward et al. (1958b), and Reinert (1959) there were some discoveries
that point in the direction of asexual embryogenesis (Levine 1950; Wiggans 1954).
Levine (1950) found that, when indole-3-acetic acid (IAA) was removed from
the culture medium, carrot callus regenerated roots and shoots. On the other hand,
Wiggans (1954) also found that the carrot cultures were able to produce plantlets
when the tissue was transferred to a medium without adenine sulfate, generating
buds first and later the roots. Thus, Levine concluded that the removal of auxin led
to root formation, followed by whole plantlets, whereas Wiggans deduced that
reducing the auxin content of the tissue led to bud formation, followed by plantlets.
Both authors concluded that decreasing the IAA in the tissues led to the formation
of plantlets through two different routes, root bud in the first case, and viceversa in
the second. Its accidental discovery opened an area of research, plenty of chal-
lenges, and wonders that have their roots and parallels in the development of plant
tissue culture.
During the 30s of the last century, several research groups used Bryophyllum
calycinum (Crassulaceae), actually known as Kalanchoë pinnata Lam. Pers., as a
model to study regeneration in plants (Freeland 1933; Howe 1931; Naylor 1932;
Yarbrough 1932, 1934). It was observed that at the edges of the leaves of
Kalanchoë appear small bipolar structures. Several species of this genus can pro-
duce somatic embryos constitutively (K. daigremontiana). Other species produce
plantlets by the action of environmental stress (K. pinnata, K. fedstchenkoi, K.
strepthantha, K. prolifera, and K. crenata) and semi constitutive plantlet-forming
species, which produce plantlets both constitutively as well as upon stress induction
(K. gastonis-bonnieri) (Garces and Sinha 2009). Some additional information of SE
in nature was generated from Tolmiea menziesii (Yarbrough 1936) and Crassula
multicava (McVeigh 1938). In all the cases, somatic embryos were found at the
edge of the leaves.
2 The History of Somatic Embryogenesis 13
SE is a case of accidental codiscovery; in the space of only 15 months, three

independent groups published seminal papers on the subject, and a fundamental
part of plant tissue culture began its spectacular development. The first of these
papers was submitted at the end of 1957 by Harry Waris’ laboratory, to the journal
Physiologia Plantarum (Miettinen and Waris 1958). Waris worked at the Botanical
Institute of the University of Helsinki. The interest of Waris was on the effect of
amino acids on the germination of various seeds. He was using amino acids such as
alanine, arginine, leucine, α-aminobutyric acid, valine, asparagine, and others. He
worked on the hypothesis that the use of different amino acids should change the
balance of the proteins synthesis during the development of the new plantlets, and
have morphogenetic consequences (Miettinen and Waris 1958; Waris 1957).
Waris found that after 3–4 months in the presence of 13.32 or 53.28 mM glycine
the original plantlets almost died. However, new green plants emerged from
“minute grains [and were] formed by some root tips of the original, morbid plant”
This report was a presentation at the meeting of the Biochemical, Biophysical and
Microbiological Society of Finland in March of 1957 (Waris 1957). Later, on
November, Waris made a presentation to the Finnish Academy of Sciences entitled
“A chemically-induced change in the morphogenesis of a flowering plant’’; this talk
was published in the Proceedings of the Finnish Academy of Science and Letters. In
this talk, Waris presented data of the effect of glycine in carrot. This effect was the
production of somatic embryos of carrot (Krikorian and Simola 1999). The next
month, and with the help of Dr. Jorma K. Miettinen, a biochemist, submitted a
paper to the journal Physiologia Plantarum entitled “A chemical study of the
neomorphosis induced by glycine in Oenanthe aquatica” (Miettinen and Waris
1958). Waris did not name the structures as “embryos” or something like this, he
used the term neomorph and neomorphosis for somatic embryo and SE, respec-
tively. During the next 4 years, Waris published other two papers related to the
neomorphosis of the genus Oenanthe. The Waris papers not only were among the
first papers in SE but also they were the first papers that documented the effect of
the nitrogen source on the morphogenesis of plant cells.
At the same time Frederick C. Steward was working at the Department of
Botany at Cornell University, on the different ways suspension cultures can grow
and multiply. He submitted two papers on June 1, 1958 and they were published
back-to-back in the American Journal of Botany (Steward et al. 1958a, b). In the
first paper, Steward et al. (1958b) reported the characterization, in particular, the
growth, and shape of freely suspended carrot, peanut, and potato cells. The second
paper describes the easy formation of roots in the liquid medium, containing
coconut milk, from the small aggregates of the suspended cells. After the roots had
been cultivated on semisolid medium, they were able to develop shoots opposite to
the position of the roots and developed into complete plantlets. Steward et al.
(1958a) documented a very ordered process from the sheath of cambium-like cells,
to the embryo-like structure. This process is reminiscent of the formation of
plantlets from zygotic embryos. Steward observed correctly the formation of vas-
cular tissue before the formation of the root and inferred that such development was
characteristic of a “proembryo.”
The third paper of this history was published by Jakob Reinert, who was
working at the Botanisches Institut der Universität in Tübingen, Germany (Reinert
1959). The paper was submitted on February 12, 1959 to the journal Planta. Reinert
was able to induce the formation of carrot shoots, by transferring the callus tissue
with roots into a white medium with several organic compounds including inositol,
choline, riboflavin, biotin, ca-pantothenate, ascorbic acid, hypoxanthine, and a
plethora of amino acids in addition to IAA and 2,4-dichlorophenoxy acetic (2,4-D).
The complexity of this “synthetic medium” was antecessor of the complete media
published in the next decade (Linsmaier and Skoog 1965; Loyola-Vargas 2012;
Murashige and Skoog 1962). Altering this synthetic medium by changing the
culture medium, Reinert was able to manipulate the formation of roots and shoots.
He deduced, correctly, that the shoots come from “adventitious bipolar embryos.”
Confirmations of these landmark papers began to appear in the literature just a
couple years later (Kato and Takeuchi 1963; Wetherell and Halperin 1963). At the
same time, the first pictures of somatic embryos were published (Kato and Takeuchi
1963; Wetherell and Halperin 1963). The source of the explants extended to Datura
innoxia anthers (Guha and Maheshwari 1964). In the following years, more species
and more different explants were used to study the SE process (Table 1.1).
However, Daucus carota, had remained the most studied, and become The Model
for the study of SE (Fujimura 2014).
Table 1.1 Summary of successful somatic embryogenesis during the first years after its discovery
Species Explant Year Reference
Oenanthe aquatica Somatic embryos 1958 Miettinen and Waris (1958),
Waris (1959)
Daucus carota Suspension cultures 1958 Steward et al. (1958a)
Daucus carota Callus 1959 Reinert (1959)
Hordeum vulgare Zygotic embryos 1961 Norstog (1961)
Cuscuta reflexa Zygotic embryos 1961, Maheshwari and Baldev (1961,
1962 1962)
Dendrophthoe falcata Zygotic embryos 1962 Johri and Bajaj (1962)
Daucus carota Callus 1963 Kato and Takeuchi (1963)
Daucus carota Callus 1963 Wetherell and Halperin (1963)
Daucus carota Seeds 1964 Steward et al. (1964)
Datura innoxia Anthers 1964 Guha and Maheshwari (1964)
Ranunculus sceIeratus Suspension cultures/stem 1965 Konar and Nataraja (1965a, b)
Nicotiana tabacum Callus 1965 Haccius and Laksmanan
(1965)
Daucus carota Callus 1966 Reinert et al. (1966)
Apium graveolens
Cichorium endivia Callus 1966 Vasil and Hildebrandt (1966a)
Petroselinurn Callus 1966 Vasil and Hildebrandt (1966b)
hortense
Solanum melongena Callus 1967 Yamada et al. (1967)
2.2 The Effect of Different Factors on the Induction

of the Somatic Embryogenesis
A set of various factors such as the genotype and physiology of the donor plant, the
explant, the composition and pH of the culture medium, the cell density, the incu-
bation conditions, and the growth regulators, among others, affect the embryogenic
response of tissues.
Three important discoveries, few years later, were: (a) the inhibitory effect on the
induction of SE by 2,4-D, (b) the necessity to dilute the cell suspension culture, and
(c) the effect of ammonium in the induction of SE (Halperin and Wetherell 1965).
When the cultures derived from root phloem grew without ammonium, they did not
form somatic embryos when the cultures were transferred to the proper conditions
for the SE induction. These calli grew very well in the presence of only nitrate, but
they were not able to form any embryogenic structure. However, the addition of
small amounts of ammonium initiated the SE process (Halperin 1966). It is known
that the amount of nitrogen in the medium is important. The relationship between
nitrate and ammonium is part of the drive force leading the process of cell differ-
entiation, in particularly SE (Fuentes-Cerda et al. 2001; Reinert et al. 1967; Tazawa
and Reinert 1969). Casein hydrolysate was also tested successfully as nitrogen
source during the induction of SE (Halperin 1995). However, there was controversy
over the role of the nitrogen source on the SE. Reinert et al. (1967) argued that
ammonium was no necessary, and that the important thing was the amount of the
nitrogen source and not the form of nitrogen. However, the same group found that
the D. carota cells cultivated in high levels of ammonium, the induction medium
for SE, contained increasing levels of this compound; the ammonium was scarcely
detected into the cells cultivated in the absence of ammonium which did not induce
SE (Tazawa and Reinert 1969). It was speculated that the intracellular ammonium
was necessary for the induction of SE. In a more systematic study carried out by
Wetherell and Dougall (1976), it was found that glutamine, glutamic acid, urea, and
alanine could, each one of them, partially replace ammonium as a supplement to
nitrate. Other explanation for the effect of ammonium was that this compound could
change the pH of the medium (Smith and Krikorian 1990). The Krikorian’s labo-
ratory (Smith and Krikorian 1990) was able to culture a D. carota proembryogenic
mass on a growth regulators-free semisolid medium containing one mM of
ammonium as the only nitrogen source. The titrated of the medium to pH 4 pro-
duced the best growth and multiplication and the culture stayed as proembryogenic
mass. If the pH was increased to 4.5 or higher the proembryos developed into
complete somatic embryos (Smith and Krikorian 1990). However, it was not
possible to discard the effect of other factors, such as the potassium present in the
medium culture (Brown et al. 1976).
Ammonium is not the only factor-driven SE. The size of the clump seems to be
important. Clusters of 20–100 cells (47–81 µm) are more suitable to produce
somatic embryos (Fujimura 2014; Halperin 1966). However, Kato and Takeuchi in
Japan (Kato and Takeuchi 1963) were able to regenerate plantlets from somatic
embryos originating from friable small callus clumps. When single cells from these
clumps were cultivated in fresh medium, no result was obtained. Nevertheless, the
presence of a clump of growing cells promotes the complete development of the
single cell into a plantlet. We know this process as a nurse culture. There is (are) a
factor or factors that are produced by the clump of cells, which is (are) necessary for
the growth of the single cell and is (are) diffused into the culture medium.
During the late 90s and earliest 20s, the laboratory of Kobayashi published a
series of papers showing that, to induce the SE in D. carota suspension cultures was
necessary to dilute the cell concentration. After several days of culture, the spent
medium contained compounds secreted by the cells. Some of these compounds,
such as the 4-hydroxybenzyl alcohol, inhibited the SE (Higashi et al. 1998;
Kobayashi et al. 1999, 2000a, b, 2001). Other compounds, with similar inhibitory
effect were found during SE induction in Larix leptolepis (Umehara et al. 2004).
These inhibitory molecules were identified as vanillyl benzyl ether (Umehara et al.
2005) and 4-[(phenylmethoxy) methyl] phenol (Umehara et al. 2007). In our lab-
oratory, we found that several phenolic compounds secreted during the induction of
SE in Coffea arabica, can inhibit the SE of several species (Nic-Can et al. 2015).
“The auxin factor” or the “auxin paradox” was known very early in the research
of the SE. Exposition of the embryogenic callus to concentrations of 2,4-D higher
than 0.45 µM did not allow the formation of the somatic embryos (Halperin 1964;
Halperin and Wetherell 1965; Reinert and Backs 1968). On the other hand, the
presence of auxin was necessary to induce the change in the genetic program of
somatic cells (Fujimura and Komamine 1979). Auxin removal was essential to
allow the induction of SE and the further development of somatic embryos
(Halperin 1964). An important problem of the production of somatic embryos is
their heterogeneity. In a callus tissue, it is possible to find a mixture of proem-
bryogenic tissue and somatic embryos at different developmental stages.
Nevertheless, it is conceivable to “synchronize” the production of somatic embryos.
An early method was the sieving of suspension cultures. The best size range to
produce synchronized cultures was between 45–75 µm (Halperin 1964). Other
factors, in addition to the size of the clumps, to produce synchronized cultures are
the species, a low ammonium content in the induction medium, and the frequent
renewing of stock culture (Fujimura 2014).
An early observation during the induction of SE was the changes in the mor-
phology of the cells that occur during the process. Most of the cultures possess two
kinds of cells: cells highly vacuolated, which in general do not divide, and
meristematic small cells. These cells are spherical, and densely packed with starch
(Quiroz-Figueroa et al. 2002). During the induction of SE, there are several
ultrastructural changes, including a remarkable increase in free ribosomal content,
decrease in endoplasmic reticulum, loss of polyribosomal aggregates, and appear-
ance of microtubules (Halperin and Jensen 1967).
2.3 Molecular Biology of Somatic Embryogenesis
Since a very rapid cell division occurred just before the formation of the globular
embryo (Fujimura and Komamine 1982), it is expected a high metabolic activity,
including the biosynthesis of macromolecules. The first papers related to the role of
protein synthesis, and transcription of genes began to appear at the beginning of the
80s. The laboratory of Raghavan published several articles related to the biosyn-
thesis of proteins and nucleic acids. They found that the rate of RNA synthesis
increased in carrot embryogenic cells following their transfer to fresh medium
(Sengupta and Raghavan 1980a). The rate of rRNA synthesis in the embryogenic
cells was lower than that in the nonembryogenic cells. However, embryogenic cells
synthesized mRNA at a higher rate than the nonembryogenic cells during the first
96 h (Fujimura and Komamine 1982; Sengupta and Raghavan 1980b). The ratio of
RNA to DNA of both cultures increased in the early stage of the culture. The ratio
increase was higher in the embryogenic tissue, suggesting that the embryogenic
culture was metabolically more active (Masuda et al. 1984) probably because the
replicon size was much reduced in the embryogenic samples (Fujimura and
Komamine 1982). The use of wheat germ system to translate the mRNA extracted
from embryogenic and nonembryogenic cells, in combination with two-
dimensional gel electrophoresis and autoradiograms, was let to determinate that
there were small, but clear differences between both systems (Fujimura and
Komamine 1982), suggesting a regulation at the transcriptional level.
The group of Dr. Z. Renee Sung at Berkeley made seminal contributions to
understand the macromolecular processes during the induction of SE. The com-
parison of the protein profile, between carrot callus and somatic embryos derived
from them, showed the presence of two proteins present only in the embryogenic
tissues (Sung 1983; Sung and Okimoto 1981). The switch to turn on and off the
biosynthesis of these two proteins was the absence or presence of 2,4-D, respec-
tively. At the beginning of these studies, it was fascinating the fact the proteic
profiles were very similar in undifferentiated and nonembryonic cells (Choi and
Sung 1984). Using antibodies against an extract of carrot somatic embryos, Choi
et al. (1987) was able to detect some proteins produced in the onset of SE. This
technique lead to the isolation of three cDNA clones, one of them codified for a
50 kDa protein that was present also in the embryogenic tissues of cassava, peach,
and maize (Choi et al. 1987).
Suspension cultures of carrot secreted considerable amounts of protein into the
culture medium, most of them glycoproteins. This secretion can depend on the
culture conditions and the initial source of the explant, among other factors. Most of
the first studies were carried out in carrot. The electrophoretic analysis of the
secreted proteins showed the presence of two glycoproteins of 65 (GP65) and 57
(GP57) kDa. The presence of 2,4-D, which inhibits the induction of SE, led to the
secretion of GP57 in both embryogenic and nonembryogenic cultures. In the
absence of 2,4-D embryogenic cultures formed somatic embryos and secreted GP65
(Satoh et al. 1986). In other study, proteins of 13, 17, 29, 38, and 46 kDa were
found in the culture medium during the induction of SE in carrot (De Vries et al.
1988). The secretion of these proteins was reduced or absent in the presence of
2,4-D. On the other hand, the presence of 2,4-D promoted the secretion of a
complete different set of proteins with molecular masses of 27, 36, 40, 44, 48, and
72 kDa. The proteins of 29, 46, 72, as well as a 52/54 kDa were high-mannose
type glycoproteins (De Vries et al. 1988). Other particular protein secreted by
embryogenic cultures was a protease inhibitor with a molecular mass of about
12.8 kDa (Carlberg et al. 1987). This protease inhibitor inhibited trypsin activity.
The inhibitor was present in both, nonembryogenic and embryogenic cultures, but
was released into the culture medium only by the embryogenic cultures (Carlberg
et al. 1987).
SE is completely blocked by inhibitors of the glycosylation, such as tunicamycin
and deoxynojirimycin. Tunicamycin treatment resulted in the presence of degly-
cosylated forms of the 46- and 52/54-kD (De Vries et al. 1988) and the inhibition of
SE. The inhibition of SE by tunicamycin can be reverted by addition of extracel-
lular proteins from untreated embryogenic cultures.
The qualitative differences between the electrophoretic protein patterns from
nonembryogenic and embryogenic cultures, have also been found in calli from
Oryza sativa (Chen and Luthe 1987) and embryogenic calli of Pisum sativum where
two proteins of 45 and 70 kDa were found (Stirn and Jacobsen 1987).
Other proteins, such as acid phosphatase or with α-mannosidase activity, were
found in the culture filtrate of both embryogenic and nonembryogenic lines,
which suggest that the release of the inhibitor from embryogenic lines was not due
to cell lysis.
2.4 Concluding Remarks
SE is a very important and interesting biological process. Scientists have been able
to carry this process from nature to the laboratory. The history of this development
shows how great discoveries are the result of multiple areas of knowledge, and the
necessity of a conceptual framework.
The basic knowledge of the SE process was initially established empirically. The
role of growth regulators, majorly auxins, was firmly settled very soon. The role of
the nitrogen source, as well as the presence of compounds that are secreted into the
culture medium, showed the importance to study all the factors possible involved in
the induction of SE. The molecular aspect of SE was later recognized, and showed
the importance of studying the biochemical and molecular aspects of the induction
of SE.
Today, it is known that there are several factors that can induce SE. Most of
these factors are related to stress (De-la-Peña et al. 2015). Among the different
kinds of stress that can induce SE are cold, heat, osmotic shock, water deficit, heavy
metals, nutrient starvation, wounding, medium culture dehydration, ultraviolet

radiation, and pH (De-la-Peña et al. 2015). The question that raises this knowledge
is: what is the common factor that leads to the induction of SE?
Acknowledgments The work from VMLV laboratory was supported by a grant received from the
National Council for Science and Technology (CONACyT, 157014).
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Chapter 3
The Many Ways of Somatic Embryo
Initiation
Attila Fehér, Dóra Bernula and Katalin Gémes
Abstract It is widely believed that all cells of a plant are totipotent and can
regenerate the whole organism. This view is supported by uncountable experi-
mental observations demonstrating the regrowth of whole plants from various
explants, even from single cells. However, recent investigations have demonstrated
that plant regeneration may proceed via transdifferentiation of meristems or root
meristem-like callus tissues due to adult stem cells present all over the plants. These
pathways do not start from single totipotent cells. There is a strong argument for
plant cell totipotency, however, and that is somatic embryogenesis. During this
process, differentiated somatic cells change their fate to develop into an embryo.
Animal embryos can develop only from the totipotent zygote and its direct
descendants (this cell state can also be artificially produced by injecting a somatic
cell nucleus into an egg cell cytoplasm during cloning). Plant cells have to be
induced to start somatic embryogenesis and not all of them are competent to
respond properly to the induction. In conclusion, plant cells cannot be considered as
totipotent per se, but some of them can regain totipotency under appropriate con-
ditions. In addition, accumulating evidence supports the view that even somatic
embryo development can follow various initial steps not necessarily requiring
cellular totipotency. Although, there are experimental observations to support the
progression of somatic embryogenesis through a zygote-like state in certain
experimental systems, in other instances the reorganization of several cells into an
embryo was described. The direct release/induction of the embryo development
program in vegetative plant cells may represent a third pathway of somatic embryo
development. In this chapter, a brief literature review is provided to support the
above view on plant cell totipotency as well as on the various ways to start somatic
embryogenesis.
A. Fehér (&) D. Bernula K. Gémes

Institute of Plant Biology, Biological Research Centre,
Hungarian Academy of Sciences, 62. Temesvári krt, Szeged 6726, Hungary
e-mail: feher.attila@brc.mta.hu
A. Fehér
Department of Plant Biology, University of Szeged, 52. Közép fasor,
Szeged 6726, Hungary

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torn this open, just like the other bean, pushing out its little white tip;
and this tip had bent down into the earth and taken a good hold
there, lengthening into a real root, and sending out little root hairs
that fastened it down still more firmly.
Fig. 89
But it was not satisfied to do all its growing below. Its upper part
now straightened itself out, and started right up into the air. From a
hoop it turned into a stem which lifted the bean clear above the earth
(Fig. 89).
This bean was no longer the round object we usually call by that
name; for its two halves had opened and spread outward, and from
between these two halves grew a pair of young leaves.
As these leaves grew larger, the two half-beans began to shrink,
growing smaller and more withered all the time (Fig. 90).
Why was this, do you suppose?
To make clear the reason of this, to show just why the two halves
of the bean grew smaller as the rest of the young bean plant grew
larger, I must go back a way.
Turn to the picture of the peony seed (Fig. 84). There you can see
how the baby plant is packed away in the midst of a quantity of baby
food. And in the picture of the morning-glory seed (Fig. 86) you see
the same thing.
You remember that day by day the baby plant ate more and more
of this food, and kept growing stronger and bigger, and that all this
time the store of food kept growing smaller and smaller.
Now, if you cut open the bean, you do not see a tiny plant set in
the midst of a store of food.
Fig. 90
Why is this? This is because the baby bean plant keeps its food in
its own leaves.
The seed coat of the bean is filled by these leaves, for each half of
the bean is really a seed leaf. In these two thick leaves is stored all
the food that is necessary to the life of the baby plant; and because
of all this food which they hold, the bean plant is able to get a better
start in life than many other young plants.
If you soak and strip off its seed coat, and pull apart the two thick
leaves, you will find a tiny pair of new leaves already started (Fig.
91); but you will see nothing of the sort in the seed of the morning-
glory, for the reason that this is not so well stored with baby food as
to be able to do more than get its seed leaves well under way.
The pea, like the bean, is so full of food, that it also is able to take
care of a second pair of leaves.
But now to go back to the young bean plant in the schoolroom
garden. We were wondering why the two halves of the bean, which
are really the first pair of leaves, kept growing thinner and smaller as
the second pair grew larger.
Fig. 91
Perhaps you guess now the reason for this. These first leaves,
called the seed leaves, feed all the rest of the young bean plant.
Of course, as they keep on doing this, they must themselves
shrink away; but they do not cease with their work till the plant is
able to take care of itself.
By this time, however, the seed leaves have nothing left to live
upon. They die of starvation, and soon fade and disappear.
So now you understand just what has happened to the leaves that
once were so fat and large.
And I hope you will remember the difference between the seed of
the morning-glory and that of the bean,—how the morning-glory
packs the baby food inside the seed, of course, but outside the baby
plant; while the bean packs it inside the two seed leaves, which are
so thick that there is no room for anything else within the seed coat.
But really, to understand all that I have been telling you, you must
see it for yourselves; you must hold in your hands the dried bean;
you must examine it, and make sure that its seed shell is filled
entirely by the baby plant; you must see it grow plump and big from
the water which it has been drinking; you must watch with sharp
eyes for that first little rip in the seed coat, and for the putting-out of
the tiny tip, which grows later into stem and root; you must notice
how the bent stem straightens out, and lifts the thick seed leaves up
into the air; and you must observe how that other pair of leaves,
which grows from between the seed leaves, becomes larger and
larger as the seed leaves grow smaller and thinner, and how, when
the little plant is able to hold its own in the world, the seed leaves die
away.
And if day by day you follow this young life, with the real wish to
discover its secret, you will begin to understand what the wise old
florist tailor meant when he said,—
“Be you young or be you old, there’s nothin’ sets you thinkin’ like a
seed.”
Fig. 92
A SCHOOLROOM GARDEN (Concluded)
T HE picture at the top of this page (Fig. 92) shows you how the
young squash plant comes into the world; for you remember that
in my pot garden I planted some squash seeds. And I hope that in
your schoolroom garden you will watch this plant as it makes its first
appearance.
The baby food of the squash vine, like that of the bean, is stored
away inside the seed leaves, which on this account are so large that
they quite fill the seed shell. They are not so thick as those of the
bean, but thick enough to hold all the nourishment that is needed to
keep the young plant alive and hearty until it is big enough to shift for
itself.
Very soon after this seed is laid in warm, moist earth, its little plant
begins to grow too large for the seed shell, and the white stem is
pushed out through the hole you notice at one end of the seed. This
stem forces its way into the earth below, and puts out a root, and
root fingers. And now its upper part begins to lengthen out and to
straighten itself. In doing this, it pulls the two seed leaves right out of
the seed coat. If it fails at once to get rid of the seed coat, it lifts this
up into the air, on top of its leaves.
Fig. 93
Often the young maple tree comes into the world in this way,
carrying its seed coat on top of its seed leaves. The maple is another
plant that packs its baby food within the seed leaves instead of round
about them. Perhaps your teacher has saved for you some maple
keys (Fig. 93), as the fruit of the maple tree is called. If you split open
a maple key, you will find hidden within one of its halves (Fig. 94) the
beautiful baby tree. This is folded away so neatly that one is tempted
to split open one key after another, for the pleasure of unpacking
other delicate baby maples (Fig. 95).
Fig. 94
But now let us find out what has happened to the peas which I
planted.
Fig. 95
Peas seem to us so much like beans, that perhaps you think the
young pea baby comes into the world in the same way as the bean
plant; but surely we have nothing here that looks at all like the bean
plant.
We see some stems having small, thin, green leaves.
Where are the fat seed leaves, filled with the baby food that keeps
the plant alive? They are not in sight, certainly, so we must start a
hunt for them.
If you will carefully remove the earth from about this little pea
plant, you will soon find that the pea seed from which it is growing
lies buried in the earth (Fig. 96). This pea seed, like that of the bean,
is made up chiefly of what really are two seed leaves, although in the
case of the pea it may seem only as a matter of politeness that we
give them the name of “leaves;” for in the pea these seed leaves lie
buried in the earth, and split open just enough to allow the little pea
plant to grow up into the air.
Fig. 96
But like the seed leaves of the bean, they are fat and full of food,
and care for the young plant just as devotedly as did those of the
bean. When this young plant needs them no more, like those of the
bean, they die of starvation.
Fig. 97
Within the acorn, the seed leaves of the great oak tree grow
together. These lie quietly in the acorn shell while sending out
supplies of food to the root and stem and leaves of the young oak
(Fig. 97). Walnut and chestnut leaves act much in the same manner.
But these first leaves of the walnut do not grow together, as you
know. Each one is packed away separately in half of the walnut
shell.
The corn has but one seed leaf, which makes it unlike all the other
plants about which we have been reading; but it resembles the pea,
the acorn, the walnut, and the chestnut in this,—that the one seed
leaf lies buried in the earth, as do their two seed leaves.
Fig. 98
The baby corn plant is very small. It does not fill the whole seed
shell, but gets its nourishment from the food by which it is
surrounded.
This picture shows you a seed or grain of corn cut in two (Fig. 98).
Of course, this is much larger than life. In the center you see the tiny
plant. All about is the baby food.
The next picture (Fig. 99) shows you the young corn plant.
Fig. 99
I want you to remember that this is the only plant we have seen
with but one seed leaf. This one seed leaf never comes out of the
seed shell. There are other plants of the same kind. All the grass
plants have but one seed leaf, and the blue flag that grows in wet
meadows, and all the lilies.
Fig.
100
Fig. 101
Only a few plants have more than two of these seed leaves. The
pine trees are among these few. This picture (Fig. 100) shows you a
baby pine tree, still cradled in its seed, surrounded by baby food; and
the next one (Fig. 101) shows you the pine just starting out in the
world, with its six seed leaves.
When you study the botany that is written for older people, you will
find that plants are set apart in separate groups, according to the
number of their seed leaves.
Strange though it may seem to you, plants with but one seed leaf
have certain habits that you will not find in a plant with two seed
leaves; and a plant with two seed leaves, long after these have
passed away, will show by root and stem that it had more than one
seed leaf.
In your schoolroom garden I should like you to grow side by side,
first a plant with but one seed leaf, next a plant with two seed leaves,
and lastly a plant with more than two.
SEEDS AS FOOD
I WANT you to think for yourselves why it is fortunate for us human

beings that many plants store away in their seeds so much baby
food.
“Because without this the little plants would die, and we should
have no new plants to make the world beautiful to live in,” some child
replies.
That answer is a good one; but it is not just the answer that I wish.
Can you think of any other way in which we all benefit by the large
supply of baby food that is packed away in certain seeds?
If the right answer to this question does not occur to you, try to
remember which of those seeds we have been reading about have
been supplied with a specially large amount of this food.
You remember that the bean holds so much baby food in its seed
leaves, that these are very fat. So do the pea, the walnut, and the
chestnut. The seed of corn, also, is well filled with baby food, only in
the corn seed it is packed around the outside of the seed leaves,
instead of the inside.
But the squash, although it puts in its seed leaves enough food to
keep its young plant well and hearty, does not lay by any great
quantity of this material. Neither does the maple tree, which also
stores the seed leaves with food, but does not fill them nearly so full
as do the bean and the pea.
And the morning-glory, which packs its precious white jelly (this is
what the little morning-glory plant likes to eat) all about its young,
lays up only just enough of this to last until the baby plant breaks out
of its seed shell.
Now, what difference do we find between these seeds?—between
the seeds of squash, morning-glory, and maple, which have only a
small supply of baby food, and those other ones, such as bean and
pea and corn and walnut and chestnut, which are packed full of
nourishment?
“Why, these last ones are good to eat!” you exclaim. “They are
part of the food we live upon, while the squash seeds, the morning-
glory seeds, and the maple seeds are not good to eat.”
Yes, that is the answer which I wished. The baby food in these
seeds makes “grown-up” food for us. We get strength and
nourishment from the same material which the mother plant has
prepared for her young.
Now try to name the different seeds which help to make up our
food. Already we have mentioned several kinds. Besides these,
there is the oat seed (from which comes oatmeal), the wheat seed
(from which comes flour for our bread), the coffee seed, the
buckwheat seed, the peanut seed, the almond seed, and many
others, some of which will come to your mind from time to time.
I wish you would make for your teacher a list of all those that you
can recall; or, better still, I should like you to collect as many as you
possibly can, and bring them to your next class. If you cannot find
the seeds just as they grow upon the plant, you may be able to get
them prepared for use. A pinch of flour, for instance, would answer
for wheat seeds, of oatmeal for oat seeds.
I have in mind a number of seeds that you can easily secure, of
which I have not spoken; and it will be interesting at our next meeting
to see which child in this class is able to make the best exhibition of
seed food.
AN IMPATIENT PLANT BABY
P LANT babies are not alike as to the time they take in finding
their way out of the ripe seed shell into the world.
Certain seeds need only two or three days in which to bring forth
their young. Perhaps we ourselves have seen the white tip of the
bean rip open its shell the second or third day after being laid upon
the moist cotton wool. But if we had not given this bean plant a good
chance to grow, it would have kept alive and hearty inside its shell
for a long time. This is not the case with all plants. Certain seeds
need to be planted soon after they are ripe. If they are not, their baby
plants die.
But usually seeds take such good care of their young, that they will
live for a long time, even if shut up in a dark closet or a table drawer,
instead of being comfortably laid away in the warm, moist earth.
Wonderful stories are told of seeds that have sprouted after having
lain buried in some Egyptian tomb for thousands of years; but the
people best fitted to judge of the truth of such stories do not believe
them. There is no doubt, however, that some seeds keep their baby
plants alive for many years.
Early in the summer the seeds of the red maple fall to the ground;
and soon after this the young plants find their way up into the world
above. Later in the year the sugar maple sheds its seeds. These lie
sleeping in the earth through the winter. When the warm spring days
come, the baby plants awake, and stretch themselves, and join the
hundreds of other, just-awakened baby plants that are flocking into
the world above. So you see that seeds of the same family have
different habits in this matter.
There is one curious tree that lives in swamps along the seashore
of hot countries. It is called the mangrove. The baby plants of this
tree are so anxious to get out into the world, that they do not wait
until the seeds in which they are hidden are set free from the
mangrove fruit.
It is as if the little plants inside the apple seed could not wait until
the apple flesh should be eaten or should decay, but insisted on
struggling first out of the seed into the apple, and then through the
apple into the light and air.
Fig.
102
This picture (Fig. 102) shows you the mangrove fruit. It looks more
like a pear than an apple. In the middle of this lies hidden one seed.
As time goes on, this grows bigger and bigger, trying to make room
for the impatient little plant within; but it does not grow fast enough to
please this ambitious young one, which finally overcomes the
difficulty by piercing the seed shell with its stem. This stem bores its
way right down through the mangrove fruit, and breaks into the outer
air. It keeps on growing in this way for many weeks, till at last it is a
foot long (Fig. 103). Try to fancy how odd a mangrove tree must look
at this time, covered with mangroves, from each one projecting this
long odd-looking beak, which one could hardly guess to be the stem
of the baby plant within the fruit.
Fig. 103
We read that these long-beaked fruits bob about with every breath
of wind in a fashion that gives the tree a still stranger look than on a
day when the air is not stirring. Picture a pear tree from every pear of
which the long stem of a baby pear tree protrudes. Would you not be
eager to find one of these pears and cut it open, and see what sort of
a baby plant it must be that could send out such a great stem?
Fig.
104
But perhaps the strangest part of the story is yet to come. At last
all of these great beaks fall away from the fruit; and from the broken
top of each grows a little bud, such as you see in the picture (Fig.
104). When this heavy beak falls upon the muddy ground below, its
pointed end strikes first, and so bores into the earth.
Even if it happens to fall into the water, it does this with so much
force that it will pierce its way to the depth of eighteen or twenty
inches, and yet remain standing erect when it strikes bottom, where
it sends out a root. When it has secured a good hold, the little bud
unfolds into four leaves. Above these grow larger, shining leaves;
and soon the ground beneath an old mangrove tree is covered with
these daring little adventurers.
A HUMPBACKED PLANT BABY
N OW let us pause for a moment, and try to recall a little of what

we have learned since school opened.
We learned that the fruit of a plant is the part which holds its seed,
and that there are many different kinds of fruits; that the burr of the
burdock, the pod of the milkweed, the puff of the dandelion, are
fruits, as well as the apple and the pear, the acorn and the walnut.
We learned that the chief importance of these fruits lies in the fact
just mentioned, that they hold the seeds of the plants.
Then we learned something about the many different kinds of
seeds, and of how these seeds managed to become separated from
the parent plant, and to get a start in life.
Next we read of the baby plant which lies hidden within every
perfect seed. We learned how this is kept safe and warm, and
supplied with food, and how at last it finds its way out of the seed
shell into the world.
If you have been using this book in the right way, not only have
you read about these things, but you have seen them with your own
eyes.
Some of the fruits you have tasted, and others you have handled.
You have examined the silky sails of the seeds which float through
the air, and the hooks and claws of those little tramps that manage to
steal free rides.
And some seeds you have planted. These you have watched day
by day, and you have seen that the baby plants burst their seed
shells much as a chick bursts its eggshell.
Now what I want you to do is this: I want you to study carefully the
different parts of these little creatures that are living out their strange,
beautiful lives under your very eyes. I want you to watch them from
day to day; to learn how they eat and drink and work and grow, until
you feel that you know them really well.
First let us look at this bean plant which is breaking its way
through the earth.
Just what do you see?
You see what looks like a thick, green hoop (Fig. 105), do you not?
What is it, this odd-looking hoop?
Fig. 105
Perhaps some of you still think that it is the root, for I remember
that I too once supposed the root was the part of the plant which first
left the bean.
But really this green hoop is made by the bent stem. We know that
this is the stem because from its lower end grows the root, while
from its upper part grow the leaves, flowers, and fruit.
Fig. 106
Certainly it is curious, the way in which this bean plant comes into
the world. Why does it not grow straight up and down, do you
suppose?—up with its stem and leaves, and down with its root?
But we must not forget that whenever some habit on the part of a
plant has filled us with surprise, sooner or later we have discovered
that the plant’s reason for it was a good one.
Fig. 107
What reason can the bean plant have for coming into the world
crooked?
Before reading further, try to think this out for yourselves. Try each
one of you to form an idea of what the bean plant gains by pushing
through the earth with this hooped stem.
I hope some of you may guess correctly. But even if you have not
been successful in naming the cause of the bean plant’s humped
back, at least you have been working your brains; and every time
you do this, you help to keep them in good order.
If you let your brain lie idle, and allow your teacher or your book to
do for you not only all the asking of questions, but also all the
thinking-out of answers, it will get as dull and rusty and good for
nothing as a machine that is laid by for a long time gets dull and
rusty and good for nothing.
I should be sorry to think that any of you children were carrying
about in your heads any such rusty, good-for-nothing brains. So if
you wish to keep them bright and clean, and in good working order,
you must try to do your own thinking.
And now, hoping you have tried to guess for yourselves the reason
of this crooked back, I will explain it to you.
But first handle carefully the tip of one of the upper leaves on the
larger bean plant. You see how delicate this is.
Then feel how firm and hard and tough is the green hoop of the
plant which is just breaking through the earth.
Now suppose the bean plant had grown straight up into the air,
would not its uppermost part have been the delicate leaf tips?

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Somatic Embryogenesis Fundamental

Aspects and Applications 1st Edition

Sustainable Finance Fundamentals 1st Edition Vargas

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Chemistry and technology of plant substances chemical

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ISBN 978-3-319-33704-3 ISBN 978-3-319-33705-0 (eBook)

Library of Congress Control Number: 2016937943

© Springer International Publishing Switzerland 2016

Printed on acid-free paper

This Springer imprint is published by Springer Nature

1 Somatic Embryogenesis. An Overview . . . . . . . . . . . . . . . . . . . . . 1

Part I Basic Aspects

9 The Relationship Between Stress and Somatic Embryogenesis . . . . 151

Part II Somatic Embryogenesis of Basic Models and Industrial

19 Somatic Embryogenesis for More Effective Breeding and

Part III Applications and Techniques

Víctor M. Loyola-Vargas and Neftalí Ochoa-Alejo

Abstract Somatic embryogenesis is one of the most powerful tools in plant

Initially, the necessity of solving important fundamental questions in plant biology,

V.M. Loyola-Vargas (&)

© Springer International Publishing Switzerland 2016 1

of micropropagated shoots to maintain plant viruses. Root cultures can be used to

1.2 Somatic Embryogenesis

1.3 Concluding Remarks

Abstract Somatic embryogenesis is used currently as a powerful tool in biotech-

Embryos, essentials for the reproduction of higher plants, were an important

V.M. Loyola-Vargas (&)

© Springer International Publishing Switzerland 2016 11

SE is a case of accidental codiscovery; in the space of only 15 months, three

2.2 The Effect of Different Factors on the Induction

2.3 Molecular Biology of Somatic Embryogenesis

2.4 Concluding Remarks

metals, nutrient starvation, wounding, medium culture dehydration, ultraviolet

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© Springer International Publishing Switzerland 2016 23

A SCHOOLROOM GARDEN (Concluded)

I WANT you to think for yourselves why it is fortunate for us human

N OW let us pause for a moment, and try to recall a little of what

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