The Use of Zygotic Embryos As Explants For in Vitro Propagation: An Overview
The Use of Zygotic Embryos As Explants For in Vitro Propagation: An Overview
The Use of Zygotic Embryos As Explants For in Vitro Propagation: An Overview
Abstract
Plant propagation in vitro via somatic embryogenesis or organogenesis is a complicated process requiring
the proper execution of several steps, which are affected by culture conditions and environment. A key
element for a successful outcome is the choice of the explants. Several studies have shown that factors
such as age, ontogenic and physiological conditions, and degree of differentiation affect the response of
the explants to culture conditions. As a general rule, younger tissues, such as zygotic embryos, are the
preferred choice for tissue culturists as they have better potential and competence to produce embryos
and organs compared to more differentiated and mature tissues. This chapter focuses on how compe-
tence and commitment to regenerate embryos and organs in cultures are acquired by somatic cells and
why zygotic embryos are so often utilized for propagation practices.
Key words: Embryo, Organogenesis, Plant growth regulators, Propagation, Somatic embryogenesis
1. Introduction
Trevor A. Thorpe and Edward C. Yeung (eds.), Plant Embryo Culture: Methods and Protocols,
Methods in Molecular Biology, vol. 710, DOI 10.1007/978-1-61737-988-8_17,
© Springer Science+Business Media, LLC 2011
229
230 Elhiti and Stasolla
1.2. Organogenesis Besides embryogenesis, plantlet formation from cultured cells can
occur through the formation of primordia, which subsequently
undergo organogenesis. In many instances, shoot primordia are
formed first followed by leafy vegetative shoots, which are then
rooted via root organogenesis. The organogenic process was first
documented by White (9) who obtained shoots from tobacco
hybrids and Nobecourt (10) who observed root formation from
carrot callus. During the following years several other plant spe-
cies were shown to form de novo shoots and roots from callus,
thanks to the finding of Skoog and Miller (11) who identified the
auxin/cytokinin balance as the main regulatory mechanism con-
trolling organogenesis. In addition to phytohormones, other
metabolites have shown to stimulate organogenesis in different
species. These metabolites include adenine, amino acids, uracil,
uridine, nicotine, and phenolic acids (12). However the interac-
tions among all these compounds are in agreement with the
notion postulated by Skoog and Miller (11); a concept which still
leads the majority of research dealing with organogenesis. An
updated review on the physiological and molecular events occur-
ring during the organogenic process is provided in (13).
This chapter provides an overview on the use of zygotic
embryos to initiate somatic embryogenesis and organogenesis
in vitro. Factors regulating embryogenic/organogenic potential
and competence are first discussed in order to appreciate why
immature and mature zygotic embryos are the preferred choices
of explants for many species.
2. The Embryo
genic/Organogenic
Pathway from
Somatic Cells Induction of embryos and/or organs from somatic cells within an
explant is a complex process, which is exemplified as consisting of
three conditions (2, 14). First, the explant must have the
“potential” to produce embryos or organs. Second, some cells
within the explant must be “competent” to respond to endoge-
nous or exogenous signals. Third, these competent cells must be
“induced” by specific signals and become “committed” to initiate
the embryogenic/organogenic pathway.
3. Morphology
and Physiology
of Embryogenic/
Organogenic Cells As indicated in the previous section, studies by Toonen et al. (24)
on carrot cultures revealed that several cell types can generate
embryos in culture, although maximal embryogenic frequency
was observed for small, highly cytoplasmic cells. Structure of
embryogenic carrot cells was further investigated by Nomura and
Komamine (23) who using fractionation studies identified single
cells (state 0) which were able to form small cellular aggregates
and developed into embryos upon removal of auxin. State 0 cells
were also small and highly cytoplasmic, thereby confirming previ-
ous observations. Accurate identification of competent cells was
rendered possible by the development of cellular markers, including
The Use of Zygotic Embryos as Explants for In Vitro Propagation 237
4. Molecular
Events Related
to Embryogenic/
Organogenic Extensive reprogramming of gene expression accompanies the
Competence transition from somatic cells into embryogenic competent cells in
response to inductive signals. Extensive effort has been focused
on the identification of “master” genes required for this transition
although it is now apparent that the induction of the embryogenic
pathway is not governed by a single gene, but it is under the
control of an intrigued genetic network. It was documented (58)
that ectopic expression of SERK resulted in a fourfold increase in
embryogenic production from Arabidopsis seedlings. The
expression of this gene, which is generally higher in cell cultures
with enhanced embryogenic capabilities (59), is unique to cells
showing a rapid response to hormonal signals and competent to
produce somatic embryos (58). Two other genes involved in the
somatic-embryogenic transition encode the transcription factors
Leafy Cotyledons 1 (LEC1) and Baby Boom (BBM) (60, 61).
Overexpression of both genes is sufficient to induce embryo
development from Arabidopsis vegetative tissue.
As suggested by Feher (2), embryogenic competence might
not be necessarily due to an induction of genetic events, but
rather to release from a suppression state. This notion is sup-
ported by studies conducted on pickle mutants in which embryos
form from root meristems. This gene encodes for a chromatin-
remodeling ATPase which is required to suppress the expression
of several embryogenesis-related genes, including LEC1, in
somatic cells (62). Therefore, embryogenic competence might be
acquired from a release of specific factors from a silencing condi-
tion mediated by the organization of chromatin (2). This notion
The Use of Zygotic Embryos as Explants for In Vitro Propagation 239
5. Zygotic Embryos
as the Preferred
Explant for In Vitro
Propagation Based on the above studies it emerges that the majority of
structural and physiological features needed for inducing somatic
embryogenesis and organogenesis in culture are present in zygotic
embryos. Zygotic embryo cells already express the “embryogenic
potential” with many of the genes required for the induction pro-
cess already expressed. Therefore, their fate is already committed
and does not need to be redirected toward a new developmental
path. This is why in many species embryogenic tissue can be
readily obtained using immature or mature zygotic embryos. Of
interest, a degree of response in culture is also related to the
developmental stage of the zygotic embryos. He et al. (64)
divided wheat embryos in several developmental stages and
showed that the higher yield of embryogenic tissue was obtained
using stage II and III embryos. A similar specificity was also
observed in conifers where immature zygotic embryos are more
responsive then their fully mature counterparts (7). Over the last
few years the number of species regenerated in culture using
somatic embryogenesis or organogenesis from zygotic embryos
has increased and includes both conifers and angiosperms
(Tables 1 and 2).
5.1. The Use Several reports describe the use of zygotic embryos as initial
of Zygotic Embryos explants for inducing somatic embryogenesis in both conifers and
for the Initiation angiosperms. In the majority of the species, the generation of
of Somatic somatic embryos comprises five steps: induction, maintenance,
Embryogenesis development, maturation, and conversion. During the induction
phase, embryogenic tissue is generated from zygotic embryos
(immature or mature), and this step usually requires high levels of
auxins and cytokinins, as well as high osmoticum. In white spruce,
for example, BA and 2,4-D are used at a concentration of 5 and
10 mM, respectively (65). These requirements are also needed
during the induction process of other species (8), although auxin
alone is often sufficient (66, 67). It is not clear which regions of
Table 1
240
Samples of species exhibiting somatic embryogenesis from zygotic embryos over the past decade
Apocynaceae Catharanthus Immature zygotic MS + 4.52 mM 2,4-D + MS + 4.52 mM MS basal salt (73)
roseus embryos 3% sucrose + 100 mg/L 2,4-D + 3% sucrose
myo-inositol + 0.4 mg/L
thiamine-HCl
Elhiti and Stasolla
arginine + 20 mg/L
praline + 0.2% activated
charcoal + 10 mM GA3
241
(continued)
Table 1
242
(continued)
sucrose + 25 mg/L commer-
cial fertilizer containing: N
10, P 52. K 10
Fagaceae Quercus robur Mature and WPM or MS + 200 mg/ WPM or MS + WPM or ½ MS + 8% (82)
immature zygotic L glutamine + 500 mg/ 0.1 mg/L BA + sucrose + 3 mg/
embryos L casein hydrolysate + 2% 0.1 mg/L IBA L ABA ® WPM or
sucrose + 2 mg/L IBA + ½ MS + 0.1 mg/L
1 mg/L BA BA + 2% sucrose
Icacinaceae Nothapodytesfo Torpedo stage of MS + 9.05 mM 2,4 D + 4.44 mM MS + 9.05 mM MS free hormones (83)
etida zygotic BA + 2.32 mM 2,4-D + 4.44 mM
embryos Kinetin + 20 g/L sucrose BA + 2.32 mM
Kinetin + 20 g/L
sucrose
Malvaceae Tilia cordata Cotyledons of MS + 0.56 mM myo-inositol + MS + 1 mM IBA ® – (84)
immature WPM vitamins + 87.6 mM MS + 131.5 mM
embryos sucrose + 4.5 mM 2,4D sucrose + 5 mM ABA
Myrtaceae Eucalyptus Mature zygotic MS + 30 g/L sucrose + 3 mg/L – MS + 30 g/L sucrose (85)
globulus embryos NAA + 100 mg/L ascorbic
acid
Meliaceae Melia Immature zygotic MS + 13.62 mM TDZ + – – (86)
azedarach embryos 3% sucrose + 2 mg/L AC
Family Species Source IM MM M/GM References
(continued)
The Use of Zygotic Embryos as Explants for In Vitro Propagation
243
Table 1
244
(continued)
Rosaceae Prunus avium Immature zygotic MS basal + 100 mg/L myo- The same induction MS basal + 100 mg/L (93)
embryos inositol + full – strength morel myo-inositol +
vitamins + 250 mg/L full – strength morel
glutamine + 2 mg/L gly- vitamins + 250 mg/L
Elhiti and Stasolla
(continued)
245
Table 2
246
(continued)
sucrose + 100 mg/L
asparagin + 100 mg/L
myo-inositol + 5 mg/L
nicotinic acid + 5 mg/L
pyrodoxine-HCl +
5 mg/L thiamine-HCl
Fabaceae Arachis hypogaea Mature zygotic MS + 30 g/L MS + 30 g/L – (103)
(Leguminosae) embryos sucrose + 4 mg/L sucrose + 0.5 mg/L
NAA + 1 mg/L BAP BAP + 0.5 mg/L
kinetin
Acacia mangium Embryo axes and MS + 9.05 mM MS + 4.55 mM MS + 10.75 mM (104)
cotyledons of 2,4-D + 13.95 mM TDZ + 1.43 mM NAA + 2.33 mM
mature zygotic KT + 100 mg/ IAA + 100 mg/ KT + 100 mg/
embryos L casein enzymatic L casein enzymatic L casein enzymatic
hydrolysate + hydrolysate + hydrolysate +
100 mg/L ascorbic 100 mg/L ascorbic 100 mg/L ascorbic
acid + 150 mg/L acid + 150 mg/L acid + 150 mg/L
glutamine + 150 mg/L glutamine + 150 mg/L glutamine +
asparagine + 150 mg/L asparagine + 150 mg/L 150 mg/L asparag-
proline + 30 g/L proline + 30 g/L ine + 150 mg/L
sucrose sucrose proline + 30 g/L
sucrose
Cajanus cajan Mature zygotic MS + 10 mM MS + 0.05 mM – (105)
embryos TDZ + 30 g/ TDZ + 30 g/
L sucrose L sucrose
Family Species Source CIM SIM RIM References
Lamiaceae Salvia sclarea Cotyledons of MS + 9.05 mM MS + 7.22 mM MS + 0.57 mM (106)
(Labiatae) immature 2,4-D + 30 g/ GA3 + 4.44 mM IAA + 0.54 mM
embryos L sucrose BA + 2.69 mM NAA + 20 g/
NAA + 30 g/L sucrose L sucrose
Oleaceae Fraxinus Mature embryos ½ MS microele- DKW + 20 g/L DKW + 20 g/L (107)
angustifolia ments + MS microele- sucrose + 4.4 mM BA sucrose + 4.4 mM
ments and BA + 0.4 mM 2,4-D
organic + 20 g/L
sucrose + 4.4 mM
BA + 0.44 mM 2,4-D
Pinaceae Larixo ccidentali Mature zygotic ½ QP + organic SH – Aqueous solution (108)
embryos (LVSH) + 2% of 0.1% IAA and IBA
sucrose + 10 mM BA
Pinus massoniana Mature zygotic – DCR + 0.5 mg/L 1/2 GD + 2% sucrose + (109)
embryos BA + 0.05 mg/L 2 mg/L IBA +
IBA + 3% sucrose 0.05 mg/L BA
Pinus strobus Embryonic TE + 5 mM IAA + 3 mM PS + 8 mM PS + 0.5 mM (110)
cotyledons and IBA + 3 mM → TDZ + 0.01 mM BA + 0.01 mM IAA
hypocotyls TE + 3 mM IAA + 6 mM IAA + 500 mg/L
BA + 6 mM TDZ + 0.4% CH + 600 mg/L
phytagel glutamine
Pinus elliottii Mature zygotic TE + 12 mM TE + 2 mM IBA + 3 mM TE + 0.01 mM (111)
embryos NAA + 15 mM BA + 9 mM IAA + 0.01 mM
2,4D + 6 mM TDZ + 30g/L IBA + 400 mg/L
2iP + 30 g/L sucrose + 500 mg/L Glutamine +
sucrose + 500 mg/L Glutamine + 500 mg/L 250 mg/
Glutamine + 500 mg/L myo-Inositol L myo-Inositol
myo-Inositol
Taxaceae Taxus wallichiana Zygotic embryos ½ WPMSH + 0.5 mg/L ½ WPMSH + 2.5 mg/L 1/5 MS (112)
BA + 1 mg/L BA + 30 g/L sucrose
The Use of Zygotic Embryos as Explants for In Vitro Propagation
2,4-D + 30 g/L sucrose
(continued)
247
Table 2
248
(continued)
5.2. The Use of Zygotic A general shoot organogenic process comprises three distinct
Embryos for Inducing steps: callus induction, shoot induction, and root induction. The
Organogenesis last two steps are collectively called regeneration.
250 Elhiti and Stasolla
6. Conclusions
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