Article Review about Somatic Embryogenesis in Plant

Induction of Somatic Embryogenesis by Binnary Expression Vector System of

BABY BOOM Gene Overexpression and Heat Shock-Inducible FRT/FLP in Calii of
Populus tomentosa Carr.


I. Introduction
Plant cell possesses remarkable developmental plasticity. One of the most intriguing
examples of this plasticity is somatic embryogenesis (SE). The SE is defined as the
developmental restructuring of differentiated somatic cells toward the embryogenic pathway
which form the basis of cellular totipotency and develop into embryo (Nishikawa et al., 2000;
Imin et al., 2004). Like their zygotic counterpart, SE has a single cell origin (Rao, 1996;
Jimenez, 2005). Direct somatic embryogenesis is a preferable pathway for plant regeneration
because a single cell origin of induced primary and secondary embryos in the most of the cases
avoids the dedifferentiated callus phase and yields genetically stable plants (Merkle, 1995).
Carrot is the first plant species in which SE was reported. Recently, induction of in vitro SE has
been shown to be successful in many plant species including angiosperms and gymnosperms.
The SE has extensively used in practical and commercial application, particularly for in
vitro clonal micropropagation (Bornman, 1993; Vasil, 1994). The SE technique allows important
practical application including germ plasma preservation, virus elimination, in vitro metabolite
production, in vitro mycorrhizal initiation, and crop improvement through cell selection, genetic
transformation, somatic hybrid, and polyploid plant production (Vicient and Martinez, 1998). SE
also provides an attractive model system for studying zygotic embryogenesis, particularly
because zygotic embryos are encased by maternal tissue and are difficult to access using
biochemical and molecular tools (Karami et al., 2009).
Process in obtaining embryo by SE is involving several steps, as illustrated in Fig. 1
(Feher, 2008). The process is initiated with somatic cells theoretically from any part of the plant.
However, substantial differences in competence are found in practice. Genetically and
developmentally determined cellular differentiation inhibits or allows the expression of
embryogenic potential depending on the genotype, developmental state and tissue type. Cells
exhibiting this potential are responsive to strong nonspecific signals such as auxin imbalance
and stress that may result in the release of the chromatin-mediated repression of embryogenic
development. The embryogenic program proceeds on a self-regulated way under permissive
physiological conditions ensuring the establishment of auxin auxotrophy and polarity (Dudits et
al., 1991). Therefore, cells which are more competent for SE are generally those coming from
young tissue (Jimenez and Thomas, 2006).

Figure 1. A hypothesis on the induction of SE in differentiated plant cells (Feher, 2008)

Genetic programs controlling embryo development in zygotic and SE display many
similarities (Middleton et al., 2007; Zhu and Perry, 2005), but the mechanisms determining the
induction phase of these two processes are different. Zygotic embryo development begins with
the formation of the zygote following fertilization, while somatic cells acquire embryogenic
competence as a result of different chemical and physical stimuli. There are now many new
molecular techniques, which will enable the dissection of these early events in the stages of
commitment and differentiation of the plant. Recent analysis of the proteome and transcriptome
have led to the identification and characterization of new genes induced SE. Understanding the
key factors promoting vegetative-to-embryogenic transition and identification of genes involved
in the induction of competence for embryogenesis and subsequent embryo development
presents a challenge for modern molecular biology (Karami et al., 2009).
II. Genes involved in expression of somatic embryogenesis
Exploration of the physiological and molecular events underlying SE is of general
interest as it may serve to improve practical applications and provide basic knowledge on the
acquisition of totipotency (Grafi and Avivi, 2004), and the regulation of the developmental
switches (Costa and Shaw, 2007). In this respect, the induction phase of SE is of primary
interest.
Several attempts have been made to identify key genes which may govern SE in
response to the induction signal (Thibaud-Nissen et al., 2003; Rose and Nolan, 2006;
Suprasana and Bapat, 2006; Tang and Newton, 2006; Legrand et al., 2007; Yazawa and
Kamada, 2007). These attempts resulted in the identification of many genes which they
expression are up- or down-regulated during embryogenesis. The change in gene expression
clearly indicate that somatic embryo induction evokes a general cellular reorganization
characterized by the expression of stress responses, the entry into the cell division cycle, the
alteration of cellular metabolism, etc (Feher et al., 2003). But, a single key gene responsible for
the induction of the developmental pathway could not identified in this approach, thus the
genetic basis for the differential regeneration capacity is still poorly understood (Feher et al.,
2008; Karami et al., 2009).
However, it has been identified that a number of genes positively influence the
regeneration competence of plant cells for SE. The SOMATIC EMBRYOGENESIS RECEPTOR
KINASE (SERK) is gene encoding signaling molecule that its overexpression enhances
embryogenic capability of the cell especially in enlarged vacuolated cells (Hecht et al., 2001;
Saze et al., 2003). Expression of the seed specific CCAAT-box binding factor LEAFY
COTYLEDON1 (LEC1) and the B3 domain transcription factor (LEC2), promote somatic embryo
formation on seedlings of Arabidopsis (Albrecht et al., 2005; Alemanno et al., 2008). The
WUSCHEL (WUS) homeodomain protein, which specifies stem cell fate in shoot and floral
meristem (Banno et al., 2001; Baudino et al., 2001), also improves somatic embryo
development in seedlings when ectopically expressed (Becker et al., 2002; Ben et al., 2004).
WUS function is not directly linked to embryo identity, but rather to the maintenance of an
undifferentiated cell state that responds to different stimuli to change the developmental fate of
tissues (Ben et al., 2004; Bender, 2004).
Similar to the AP2/ERF family of transcription factors, BABY BOOM (BBM) has been
reported to be expressed preferentially in developing embryos and seeds (Singh and Bhalla,
2006). AP2/ERF proteins are characterized by the presence of AP2/ERF DNA binding domain
that is thought to mediate DNA binding and proteinprotein interaction (Blilou et al., 2005).
Ectopic expression of BBM in Arabidopsis and Brassica led to the spontaneous formation of
somatic embryos and cotyledon-like structures on seedlings. Ectopic BBM expression induced
additional pleiotropic phenotypes, including neoplastic growth, hormone-free regeneration of
explants, and alterations in leaf and flower morphology (Singh and Bhalla, 2006). In tobacco
heterologous BBM expression induced spontaneous shoot and callus formation, while a
cytokinin pulse was required for somatic embryo formation (Boutilier et al., 2002). One major
drawback in using the BBM gene is that transgenic plants exhibit additional pleiotropic
phenotypes due to the continuous expression of the BBM gene (Singh and Bhalla, 2006).
III. Molecular approach for SE induction involving of BBM gene overexpression
Strategies for removing marker genes or short spacer sequences from host plants using
site-specific recombinases have been developed (Caliscan et al., 2004; Campbell et al., 1998;
Cao, 2003; Casson et al., 2005). Site-specific recombination systems function through
interactions of a recombinase with its specific recognition sites (Braybrook et al., 2006).
Recombinase-mediated excision between directly orientated recognition sites results in the
removal of the intervening DNA, leaving one recognition site intact (Caliscan and Cuming,
1998). More recently, a novel technology, GM-gene-deletor, based on bacteriophage Cre/loxP
and Saccharomyces cereviceae FLP/FRT site-specific recombination systems was developed
to automatically eliminated all functional foreign genes with a high efficiency from pollen and
seeds of transgenic tobacco (Chang, 2003).
Previous studies have demonstrated that overexpression of BBM in Arabidopsis and
Brassica led to the spontaneous formation of somatic embryos (Singh and Bhalla, 2006). In a
research by Deng et al. (2009), a molecular approach for induction of plant regeneration via
somatic embryogenesis based on BBM gene overexpression and heat shock-inducible
FRT/FLP system was introduced by using Chinese white poplar (Populus tomentosa Carr.) as
model plant. Schematic representation of T-DNA used for plant transformation is shown in Fig.
2 (Deng et al., 2009).


Figure 2. Schematic representation of (A) T-DNA region of vector pLFFLPBBM and (B) the expected T-
DNA fragment following excision (Deng et al., 2009)

In this vector, BBM gene under the control of CaMV 35S promoter was used to induce
the formation of somatic embryos and plant regeneration. However, it has demonstrated that
BBM overexpression also leads to morphological changes in transgenic plants (Singh and
Bhalla, 2006). In pLFFLPBBM vector, two FRT sequences as the recognition sites flanked the
sequences including FLP recombinase gene and BBM gene. The heat shock promoter
HSP18.2, which showed no detectable expression in Arabidopsis under normal conditions
except for weak expression in secondary root hairs and could be induced strongly in the
presence of heat stress (Chapman et al., 2000; Che et al., 2006), was fused to FLP gene to
control removal of BBM gene. Theoretically, heat shock induced expression of FLP gene would
lead to the elimination of all sequences between the two FRT sites. Therefore, it is expected
that the morphological changes caused by overexpression of BBM gene would be eliminated by
heat shock treatment after plant regeneration via somatic embryogenesis.
The induction of somatic embryogenesis was conducted by infecting 21 calli of Chinese
white poplar (Populus tomentosa Carr.) with A. tumefaciens harbouring pLFFLPBBM, and
cultured on hormone free solid WPM medium. As shown in Fig. 3 and summarized in Table 1,
no somatic embryos were formed on non-transformed calli, while 12 somatic embryos were
induced from the 6 transgenic calli approximately 4 weeks after infection. Out of the 12 somatic
embryos, 6 embryos germinated to form plantlets on 1/2 strength WPM medium.

Table 1. Poplar regeneration via somatic embryogenesis based on BBM gene overexpression and heat
shock-inducible excision of transgenes (Deng et al., 2009)


This result is generally consistent with the previous research findings, in which ectopic
expression of the BBM gene in Arabidopsis primarily induced spontaneous somatic embryo
formation from the seedlings (Bhalla and Singh, 2006). In tobacco, heterologous BBM
expression induced spontaneous shoot and callus formation (Boutilier et al., 2002). In this study,
the six putative transgenic plants showed phenotypic alterations, including dwarf phenotype,
and small wrinkled leaves after culturing for another 60 days as shown in Fig. 3. This result in
accordance with study by Boutilier et al. (2002) that demonstrated that the transgenic
Arabidopsis plants overexpressing the BBM gene were dwarf to medium in size and produced
wrinkled leaves. These plants grew very slowly and were slow to flower, but were completely
fertile.

Figure 3. Induction of somatic embryogenesis and plan regeneration. (A) No somatic embryos induced
from the non-transformed callus. (B) Somatic embryos induced from the transformed callus that indicate
by arrow point. (C) regenerated plant derived from somatic embryos (1) without heat shock treatment and
(1T) with heat shock treatment at scale bar = 40 cm (Deng et al., 2009)

Heat shock treatment of transgenic plant was performed to eliminate the morphological
changes caused by continuous expression of BBM gene. But among 6 transgenic lines (1T-6T)
subjected to heat shock treatment, only 4 transgenic lines developed into phenotypically normal
plants within 56 weeks of growth, whereas 2 transgenic lines (4T and 6T) still exhibited the
dwarf phenotype. Southern blot analysis verified that BBM gene as hybridizing bands present in
2 transgenic plants (4T and 6T) with dwarf phenotype even after heat shock treatment, as
shown in Fig. 4. During the Southern blot analysis, genomic DNA digested with EcoR1 which
has no enzyme site in the plasmid, the radio-labelled BBM cDNA was used as a probe in the
hybridization experiments, and pLFFLPBBM plasmid was used as positive control.

Figure 4. Southern blotting analysis of regenerated plant. Lanes 1-6 for regenerated plants without heat
shock treatment, lanes 1T-6T for regenerated plants with heat shock treatment, WT for wild type plants, P
for positive control of pLFFLPBBM plasmid.
These results demonstrated that the heat inducible FRT/FLP system successfully
excised the BBM gene in transgenic lines with excision efficiency at 66.7%. Similar results on
variation in excision efficiency have been reported previously (Caliscan and Cuming, 1998;
Caliscan et al., 2004; Chang, 2003; Du and Chen, 2000; Dumas et al., 1995). Presumably
variations in excision efficiency were caused by chromosomal position of the transgenic loci in
and/or the difference in recombinase activity in plants (Dunwell, 1998). In this study, the FLP
excision system driven by the HSP18.2 promoter was able to remove the transgene, resulting in
generation of the normal plants.
Previous researches demonstrated heat shock promoters were reliable in providing a
conditional expression of the gene (Dunwell et al., 2004; Edward et al., 2000). Heat shock
promoters can work properly in heterologous systems since the response mechanisms seem to
be evolutionarily conserved in many organisms (Egertsdotter and von Arnold, 1995). Heat
inducible expression of a site-specific recombinase can indeed provide a universal genetic
switch for the genome manipulation (Eshed et al., 1999; Fambrini et al., 2006). Heat inducible
FRT/FLP system can be used to separate a gene of interest from its promoter by a block
fragment flanked by FRT target sites to silence the gene of interest.
IV. Conclusion
Somatic embryogenesis considered as an attractive issue because it provide a tool for
massive propagation of commercial crops and as a potential model system for the study of the
regulation of gene expression required for the earliest developmental events in the life of higher
plants (228). As initial basis for cellular and genetic engineering, SE plays an important role in
genetic transformation, somatic hybridization, and somaclonal variation.
Even the genetic basis for the differential regeneration capacity is still poorly understood,
a number of genes that positively influence the regeneration competence of plant cells for SE
have been identified and available for molecular application such as for design of vector system
to induce SE in plant. For example, Deng et al. (2009) has provide a binary expression vector to
induce somatic embryogenesis in calii of Chinese white poplar (Populus tomentosa Carr) based
on overexpression of BBM gene from Arabidopsis plant. This novel method provides new insight
into development SE application and further research on optimization on process condition may
need to increase efficiency of transformation and gene excision.
Application of heat induced FRT recombinase activity to control gene expression by
deletes block fragment and switches on expression of the gene of interest also considered as
interesting in development of transgenic plants. Heat-inducible expression of the site-specific
recombinase system can also be used to remove the selectable marker gene, resulting in
marker-free transgenic plants.
V. Selected Reference
Boutilier K, Offringa, R., Sharma, V.K., Kieft, H., Oullet, T., Zhang, L., Hattori, J., Liu, C.M.,
Lammeren, A.A., Miki, B.L., Custer, J.B., and Lookeren-Campagne, M.M., 2002,
Ectopic Expression of BABY BOOM Triggers a Conversion from Vegetative to
Embryonic Growth, Plant Cell, 14, 1737-1749.
Braybrook, S.A., Stone, S.L., Park, S., Bui, A.Q., Le, B.H., Fischer, R.L., Goldberg, R.B., and
Harada, J.J., 2006, Genes Directly Regulated by LEAFY COTYLEDN2 Provide Insight
into the Control of Embryo Maturation and Somatic Embryogenesis, Proc. Natl. Acad.
Sci. USA., 103, 3468-3473.
Costa, S., and Shaw, P., 2007, Open Minded cells: How Cells can Change Fate, Trends Cell.
Biol., 17, 101-106.
Deng, W., Luo, K, Li, Z., and Yang, Y., 2009, A Novel Method for Induction of Plant
Regeneration via Somatic Embryaogenesis, Plant Science, 177, 43-48.
Feher, A., Pasternak, T.P., and Dudits, D., 2003, Transition of Somatic Plant Cells to an
Embryonic State, Plant Cell, Tissue and Organ Culture, 74, 201-228.
Feher, A., 2008, The Initiation Phase of Somatic Embryogenesis: What We Know and What we
Dont, Acta Biologica Szegediensis, 52(1), 53-56.
Hecht, V., Vielle-Calzada, J.P., Hartog, M.V., Schmidt, D.L., Boutilier, K., Grossniklaus, U., and
de Vries, S.C., 2001, The Arabidopsis Somatic Embryogenesis Receptor Kinase 1
Gene is Expressed in Developing Ovules and Embryos and Enhances Embryogenic
Competence in Culture, Plant Physiol., 127(3), 803-816.
Jimenez, V.M., 2005, Involvement of Plant Hormones and Plant Growth Regulators on in vitro
Somatic Embryogenesis, Plant Growth Regulation, 47, 91-110.
Karami, O., Aghayaisi, B., and Pour, A.M., 2009, Molecular Aspects of Somatic-to-Embryonic
Transition in Plants, J. Chem. Biol., 2, 177-190.
Singh, M.B., and Bhalla, P.L., 2006, Plant Stem cells Carve Their Own Niche, Trend in Plant
Science, 11(5), 1360-1385.
Vicient, C.M., and Martinez, F.X., 1998, The Potential Uses of Somatic Embryogenesis in
Agroforestry are not Limited to Synthetic Seed Technology, Revista Brasileira de
Fisiologia Vegetal, 10(1): 1-12.