Plant Cell Tiss Organ Cult (2007) 90:18 DOI 10.

1007/s11240-007-9249-9

REVIEW

Acquisition of embryogenic competence during somatic embryogenesis

Parameswari Namasivayam

Received: 19 April 2007 / Accepted: 21 May 2007 / Published online: 4 July 2007 Springer Science+Business Media B.V. 2007

Abstract This review focuses on investigation in acquisition of embryogenic competence during somatic embryogenesis in the last ve decades. In tissue culture, differentiated somatic cells acquire embryogenic competence and proliferate as embryogenic cells during the induction phase. These embryogenic cells are important because they differentiate to form somatic embryos at a later time. Various molecular and structural markers for detecting embryogenic cells or enhancing embryogenic competence are summarized and implications of the ndings are discussed. Keywords Embryogenic competence Somatic embryogenesis Structural markers Molecular markers Induction

Introduction Somatic embryogenesis is one form of asexual reproduction starting from isolated somatic or gametic (microspore) cells (Zimmerman 1993)

P. Namasivayam (&) Department of Cell and Molecular Biology, Faculty of Biotechnology and Biomolecular Sciences, University Putra Malaysia (UPM), 43400 Serdang, Selangor, Malaysia e-mail: parameswari@biotech.upm.edu.my

whereby these cells under favourable experimental condition are induced to form a somatic embryo in vitro. This is a remarkable phenomenon unique to plants only. The process is feasible because plants possess cellular totipotency whereby individual somatic cells can regenerate into a whole plant (Reinert 1959). Since most somatic cells are not naturally embryogenic, an induction phase is required for the cells to acquire embryogenic competence (Dodeman et al. 1997). This is in contrast to the zygote in sexual reproduction which is intrinsically embryogenic. Nevertheless, spontaneous in vivo somatic embryogenesis does occur in some species. For example, in Bryophyllum (Yarbrough 1932) and Malaxis (Taylor 1967), adventitious buds can develop naturally on leaf margins without induction. In general, the somatic embryogenesis process can be divided into two phases: induction and expression. During the induction phase, differentiated somatic cells acquire embryogenic competence and proliferate as embryogenic cells. In the expression phase, the embryogenic cells display their embryogenic competence and differentiate to form somatic embryos (Jimenez 2001). The two phases were suggested to be independent of each other and inuenced by different factors (Jimenez 2001). The term embryogenic cells is restricted to those cells that have completed their transition from a somatic state to one in which no further exogenous stimuli, such as the application of growth regulators, are necessary to produce the somatic embryo (Komamine et al. 1992; de Jong

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et al. 1993). The cells in a transitional state that still require only minimal exogenously applied stimuli to become embryogenic are dened as competent cells (Toonen et al. 1994). It is still unclear what changes a somatic cell has to undergo in order to become an embryogenic cell and capable of forming an embryo at a later stage of development. In general, an embryoid may arise from a single cell, or a group of cells budding, depending on neighbour relationships of cells within the explant (Williams and Maheswaran 1986).

endogenous hormone levels in the explant (Jimenez 2001). The range of possible induction treatments suggests that it is unlikely that a single inducing molecule is responsible (Toonen and de Vries 1996) for embryogenic competence.

Acquisition of embryogenic competence Although acquisition of embryogenic competence was investigated for many years, the emphasis was on comparative morphology and the analysis of cell division patterns during the early stages of embryo development (Halperin 1966; Halperin and Jensen 1967; Ho and Vasil 1983; Karlsson and Vasil 1986; Jones and Rost 1989). Studies using light and electron microscopy have provided detailed descriptions of the morphological and cellular changes that characterise embryogenic competence (Fransz and Schel 1991a; Dubois et al. 1991; Guzzo et al. 1994; Garrido et al. 1995; Pedroso and Pais 1995) and embryonic development (Rohr et al. 1989; Fransz and Schel 1991b; Canhoto et al. 1996; Jasik et al. 1995; Puigderrajols et al. 2001). Based on histological observations on various plant systems such as carrot (Halperin and Jansen 1967), sugarcane (Ho and Vasil 1983), alfalfa (Dos Santos et al. 1983), pearl millet (Taylor and Vasil 1996), and cork oak (Puigderrajols et al. 2001), the embryogenic cells that form somatic embryos are characterised generally as small, isodiametric in shape, have large and densely staining nuclei and nucleoli, and are densely cytoplasmic. It is not at all clear how the embryogenic cells originate. Later, the whole developmental sequence from single cells to heart shaped embryos was fully documented for carrot cell suspension culture by video cell tracking (Toonen et al. 1994; Yasuda et al. 2000). This study revealed that oval, elongated and spherical cells were capable of developing into somatic embryos with varying frequency (Toonen et al. 1994). Whether the differential ability of somatic cells to become embryogenic reects differential ability of different genotypes or of different cell types within the same genotype is not clear (De Jong et al. 1993). In another approach, the somatic embryogenesis receptor kinase (Serk) gene, was used as a molecular marker for embryogenic competence to track the development of somatic embryos in carrot culture (Schmidt et al. 1997). In this case, a class of

Induction of somatic embryogenesis Explant cells can be induced to go through a direct or indirect somatic embryogenesis by modulating tissue culture conditions. In direct somatic embryogenesis, embryos develop directly on the surface of organised tissue such as a leaf, stem segment, zygotic embryo, young inorescence, from protoplasts and from microspores (Williams and Maheswaran 1986). Alternatively, indirect somatic embryogenesis can occur in which there is an intermediary step of callus formation or cell suspension culture. In these cases, a more complex medium with additional factors is required to induce dedifferentiation and initiation of cell division in the explant before they can express embryogenic competence (Williams and Maheswaran 1986). The conditions for the induction of somatic embryogenesis in different species and cultivars are usually discovered by trial and error (Jacobsen 1991; Henry et al. 1994) by analysing effects of different culture conditions such as: plant growth regulator balance, osmotic conditions, changing pH, amino acid and salt concentrations, heat shock and treatment with various chemical substances (Ammirato 1983; Armstrong and Green 1985; Rhodes et al. 1986). Other than auxin as the main inducer (Feher et al. 2002), there have been reports for somatic embryogenesis in response to the presence of other growth regulators such as cytokinin (Sagare et al. 2000) or abscisic acid (ABA) (Bell et al. 1993; Nishiwaki et al. 2000) and also in the absence of growth regulators (Choi et al. 1998). The frequency of induction of this process does not only depend on culture conditions, but also on the particular genotype, tissue and stage of development of the explant material (Carman 1990), as well as

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elongated cells on the explant surface was reported to be competent for embryogenesis. On the other hand, when the same approach was employed for Dactylis glomerata leaf explants, small isodiametric cells rich in cytoplasm were identied as cells competent to form embryos (Somleva et al. 2000). All these studies showed that competent cells have a variable appearance that prevents their identication on the basis of morphology. The initial studies on carrot and alfalfa cultures reported that cell polarity (Smith and Krikorian 1990a; Dijak et al. 1986) and an asymmetric rst cell division (Komamine et al. 1990; Bogre et al. 1990) are involved in the initiation of somatic embryogenesis. Exogenous growth regulators probably modify the cell polarity by interfering with pH gradients or electrical elds around the cells (Smith and Krikorian 1990a; Dijak et al. 1986). However, the cell tracking system developed by Toonen et al. (1994) demonstrated that members of the heterogenous embryogenic carrot cell population were capable of forming somatic embryos after either apparently equal or unequal rst cell division. This implies that the correct plane of division is not necessary for somatic embryogenesis. However, asymmetrical distribution of intracellular molecules or cell constituents correlates with differential cell fate (Vroemen et al. 1999). For example, McCabe et al. (1997) observed that cell wall antigen on cells destined to form embryos segregates asymmetrically during a formative division, producing one daughter cell with a cell wall antigen recognized by the antibody JIM8 and the other without. The epitopefree cells ultimately formed somatic embryos and the labelled daughter cell died (McCabe et al. 1997). Several studies have showed that changes in intracellular pH may also contribute to the acquisition of embryogenic competence (Schaefer 1985; Smith and Krikorian 1990a,b). For example, in alfalfa leaf protoplast derived cells, cytoplasmic and vacuolar alkalinization and medium acidication were shown to be correlated with the activation of cell division (Pasternak et al. 2002). They also reported that the cytoplasm-rich embryogenic cells had a tendency to exhibit higher vacuolar pH values in comparison to the non-embryogenic vacuolated cells. Another feature for the acquisition of embryogenic competence is physical isolation of the cell from others such as absence of plasmodesmatal contact

with the neighbouring cells in the maize embryogenic culture (Fransz and Schel 1991a), and deposition of callosic wall in the embryogenic cells of Cichorium (Dubois et al. 1990), Trifolium (Maheswaran and Williams 1985) and coconut (Verdeil et al. 2001). More recently, a unique ultrastructural feature designated the extracellular matrix (ECM) was observed in Brassica napus during acquisition of embryogenic competence (Namasivayam et al. 2006). The ECM was reported to be present on the surface of embryogenic tissues, but not in the non-embryogenic tissues. The ECM layer was found to be dominant in the pre-embryogenic stage and reduced to fragments during embryo growth and development in mature embryogenic tissue. These studies suggest that isolation may be required to allow induction of new morphogenetic events and to prevent interference from adjacent tissues which are degenerating or committed to different pathways (Maheswaran and Williams 1985). Interestingly, the zygote develops in cytoplasmic isolation too. Also, secreted proteins such as arabinogalactan proteins (AGPs) have been demonstrated to promote embryogenesis in suspension cultures of carrot (Kreuger and van Holst 1993, 1995; Toonen et al. 1997; Van Hengel et al. 2001) and in Norway spruce (Egertsdotter and van Arnold 1995). In another example, carrot EP3 endochitinase proteins (Kragh et al. 1996) secreted into the culture medium, which were able to rescue somatic embryo development in the temperature sensitive mutant ts11 (De Jong et al. 1992, 1995) and also promoted somatic embryogenesis in wild type carrot protoplasts (Van Hengel et al. 2001).

Genes involved in the acquisition of embryogenic competence The process of acquisition of embryogenic competence by somatic cells must involve reprogramming of gene expression patterns as well as changes in the morphology, physiology, and metabolism. These alterations reect dedifferentiation, activation of cell division and a change in cell fate. Also, the changes are dependent on the down regulation of some functioning genes in differentiated cells and up regulation of genes which are required for the transition to happen.

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Of all the genes that have been isolated during somatic embryogenesis, only one, somatic embryogenesis receptor-like kinase (Serk), has successfully been shown to be a specic marker distinguishing individual embryo-forming cells in carrot suspension cultures (Schmidt et al. 1997). The Serk gene was found to be expressed during proembryogenic mass formation and up to the globular stage during carrot somatic embryogenesis (Schmidt et al. 1997). It could also be detected in zygotic embryos up to the early globular stage, but not in unpollinated owers or any other tissue. Cell tracking experiments using a Serk promoter::luciferase reporter construct veried that somatic embryos were indeed derived from SERKexpressing cells (Schmidt et al. 1997). Later, an Arabidopsis thaliana homologue (AtSerk1) of the carrot Serk cDNA was cloned as one of the ve members of a small gene family (Hecht et al. 2001). The AtSerk1 was shown to be expressed in developing ovules by in situ hybridisation (Hecht et al. 2001). In mature ovules, expression was restricted to the embryo sac where it was expressed in all cells. Following fertilization, AtSerk was expressed in the endosperm and the zygote, and in embryos through the heart stage, at which time expression ceased. A low level of expression was also detected in adult vascular tissues. AtSerk1 gene expression was also observed in the shoot apical meristem (SAM) and cotyledons of Arabidopsis seedlings treated with auxin. These are the sites at which embryogenic callus emerges in Arabidopsis (Mordhorst et al. 1998; von Recklinghausen et al. 2000). These observations indicated that AtSerk1 expression was not restricted to embryogenic cells, but was characteristic of those cells capable of responding to hormone signals and competent to form somatic embryos or embryogenic cell cultures (Hecht et al. 2001). Ectopic expression of AtSerk1 under control of the strong constitutive 35S promoter (35S:AtSerk1), from the Cauliower Mosaic Virus (CaMV), showed that the frequency of the initiation of somatic embryos was increased by approximately 4-fold in transgenic seedlings (Hecht et al. 2001), implying AtSERK1 increases embryogenic competence. In addition to genes whose ectopic expression induces embryo development, loss-of-function mutants in which embryogenesis is promoted have also been isolated. Mordhorst et al. (1998) reported

that some of the Arabidopsis mutants such as primordia timing ( pt ) [allelic to constitutive photomorphogenic2 (cop2) (Hou et al. 1993), altered meristem program 1(amp1) (Chaudhury et al. 1993), ha uptling (hpt) (Jurgens et al. 1991)], clavata1 (clv1), clavata3 (clv3) and pickle (pkl) (Ogas et al. 1997) showed an increased frequency of somatic embryogenesis compared to the wild type. In these cases, somatic embryo development was induced by culture of either immature embryos or germination of seeds in liquid media containing 2,4-D (Mordhorst et al. 1998, 2002; von Recklinghausen et al. 2000). Somatic embryos originated from the SAM and cotyledon axils of the germinating seedlings. They proposed that the enhanced embryogenic capacity of these mutants is an indirect effect, resulting from an increased number of undifferentiated cells in the SAM of these mutants. Hecht et al. (2001) reported that expression of AtSERK1 was higher in cell cultures derived from the amp1 mutant of Arabidopsis compared to the wild type or calli derived from 35S::AtSERK1 plants. Hecht and colleagues (2001) hypothesized that one of the effects of AMP1 activity could be to suppress the expression of AtSERK1 after germination. This is supported by their earlier ndings that loss of function of AMP1 had a stronger effect on embryogenic potential and plant development than 35S promoterdriven AtSERK1 expression. AMP1 has been cloned recently and found to encode a putative glutamate carboxypeptidase that may have a role modulating levels of a signalling molecule involved in meristem size (Helliwell et al. 2001). Induction of embryo development can also occur on leaves of Arabidopsis plants ectopically expressing Leafy cotyledon 1 (LEC1; Lotan et al. 1998) or LEC2 (Stone et al. 2001) and on roots in the pickle (pkl) mutant (Ogas et al. 1997, 1999). LEC1 encodes a transcription factor homologue, the CCAAT boxbinding factor HAP3 subunit (Lotan et al. 1998). LEC1 is required for the specication of cotyledon identity and the completion of embryo maturation in Arabidopsis (Meinke 1992; West et al. 1994). Ectopic postembryonic expression of LEC1 in vegetative cells of Arabidopsis induces the expression of embryospecic genes and initiates formation of embryo-like structures (Lotan et al. 1998). The pattern of LEC1 gene expression strongly suggests that LEC1 functions specically during embryogenesis, including the earliest embryonic period (Lotan et al. 1998). The loss

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of function mutant lec1 shows trichome development on cotyledons, suggesting that early vegetative development is occurring during late embryogenesis (Meinke et al. 1992; West et al. 1994). Lotan and colleagues (1998) suggested that LEC1 might be involved in inducing and maintaining embryogenesis while suppressing vegetative development. Recently, roots of the pickle (pkl) mutant were shown to retain some embryonic characteristics such as the synthesis of particular seed-type fatty acids. After removal from the mutant plant and transfer to growth regulator-free medium, pkl roots spontaneously produced embryolike structures (Ogas et al. 1997). Pkl encodes a putative CHD3 protein, a chromatin remodelling factor proposed to act as a negative regulator of a transcription (Ogas et al. 1999). They found that the LEC1 was expressed in pickle roots as well as in pkl seeds before germination, suggesting PKL is necessary for repression of LEC1 transcripts to regulate developmental transition from embryogenic to vegetative state. This was further supported by microarray studies during germination which showed that Pkl expression is necessary for repression of some regulators of embryogenesis including the LEC genes (Rider et al. 2003). Using subtractive hybridisation, a differentially expressed cDNA was identied during Brassica napus microspore embryogenesis, and the gene named Babyboom (Bbm) (Boutilier et al. 2002). The Bbm gene codes for an Apetala 2-domain transcription factor, and will generate somatic embryos on transgenic Arabidopsis cotyledons when ectopically over-expressed (Boutilier et al. 2002). This raises the possibility that BBM may be involved in enhancing embryogenic competence during somatic embryogenesis. Ectopic expression of Bbm in Arabidopsis also induced the expression of Histone deacetylase genes (Hd2A, Hd2B and Hd2C) in pre-embryonic tissues and somatic embryos, suggesting that Hd2 gene expression may be associated with the competence of tissues to undergo somatic embryogenesis (Zhou et al. 2004). Previously, Arabidopsis HD2 proteins have been demonstrated to repress transcription by interacting with plant transcription factors in vivo (Wu et al. 2000, 2003). Over-expression of HD2A protein in transgenic Arabidopsis plants generated pleiotropic developmental abnormalities including aborted seed development (Zhou et al. 2004). It was suggested that normal expression of histone deacetylases is essential for normal plant

development which includes regulation of gene expression during embryogenesis. Zuo et al. (2002) used a chemically inducible activation tagging system to identify genes whose over-expression induces the formation of somatic embryos on Arabidopsis tissues without the need for external hormonal treatments. The identied allele, Plant growth activator 6 (Pga6), was found to be identical to WUSCHEL (WUS), encoding a homeodomain protein previously shown to be involved in specifying stem cell fate in shoot and oral meristems (Laux et al. 1996). WUS/PGA6 is presumably involved in promoting vegetative to embryogenic transition and/or maintaining the identity of the embryonic stem cells.

Summary In spite of almost half a century of research carried out in the area of plant somatic embryogenesis, knowledge on acquisition of somatic embryogenesis is still in its infancy. Although earlier work assumed that all plant cells were equally totipotent, recent evidence suggests that only a subset of embryogenic cells can be developed into embryos. Some researchers have reported morphological and cellular differences that indicate acquisition of embryogenic competence for some plant species. However, there is not a universal structural or physiological marker that is potentially applicable to various plant species to identify competent or embryogenic cells that are capable of forming an embryo at a later time. Partly, this could be due to inefcient experimental techniques and lack of an experimental system with a well-dened transition phase between somatic and embryogenic cell types. However, with advancements in the eld of molecular biology and tissue culture, improvements have been made to tissue culture systems and more progress in isolating genes involved in the induction phase has been reported. Then again, most of the identied genes may not play a direct role in the induction of somatic embryogenesis, except for the Serk gene. In spite of now having Serk as a molecular marker for competent cells, we are still far from understanding the key events underlying the transition of differentiated somatic cells to the totipotent and embryogenic cell state. Reverse and forward genetics studies have also shown that there are several genes which when over-expressed or

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Plant Cell Tiss Organ Cult (2007) 90:18 Dijak M, Smith DL, Wilson TJ, Brown DCW (1986) Stimulation of direct embryogenesis from mesophyll protoplasts of Medicago sativa. Plant Cell Rep 5: 468470 Dodeman VL, Ducreux G, Kreis M (1997) Zygotic embryogenesis versus somatic embryogenesis. J Exp Bot 48:14931509 Dos Santos AVP, Cutter EG, Davey MR (1983) Origin and development of somatic embryos in Medicago sativa L. (alfalfa). Protoplasma 117:107115 Dubois T, Guedira M, Dubois J, Vasseur J (1990) Direct somatic embryogenesis in roots of Cichorium: is callose an early marker? Ann Bot 65:539545 Dubois T, Guedira M, Dubois J, Vasseur J (1991) Direct somatic embryogenesis in leaves of Cichorium: a histological and SEM study of early stages. Protoplasma 162:120127 Egertsdotter U, von Arnold S (1995) Importance of arabinogalactan proteins for the development of somatic embryos of Norway spruce (Picea abies). Physiol plantarum 93:334345 Feher A, Pasternak T, Otvos K, Miskolczi P, Dudits D (2002) Induction of embryogenic competence in somatic plant cells: a review. Biologia 57:512 Fransz PF, Schel JHN (1991a) Cytodifferentiation during the development of friable embryogenic callus of maize (Zea mays) Can J Bot 69:2633 Fransz PF, Schel JHN (1991b) An ultrastructure study on the early development of Zea mays somatic embryos. Can J Bot 69:858865 Garrido D, Vicente O, Heberle-Bors E, Rodriguez-Garcia MI (1995) Cellular changes during the acquisition of embryogenic potential in isolated pollen grains of Nicotiana tabacum. Protoplasma 186:220230 Guzzo F, Baldan B, Mariani P, Loschiavo F, Terzi M (1994). Studies on the origin of totipotent cells in explants of Daucus carota L. J Exp Bot 45:14271432 Halperin W, Jensen WA (1967) Ultrastructural changes during growth and embryogenesis in carrot cell cultures. J Ultrastruct Res 18:428443 Halperin W (1966) Alternative morphogenetic events in cell suspensions. Amer J Bot 53:443453 Hecht V, Viella-Calzada J-P, Hartog MV, Schmidt Ed DL, Boutilier K, Grossniklaus U, de Vries SC (2001) The Arabidopsis somatic embryogenesis receptor kinase 1 gene is expressed in developing ovules and embryos and enhances embryogenic competence in culture. Plant Physiol 127:803816 Helliwell CA, Chin-Atkins AN, Wilson IW, Chapple R, Dennis ES, Chaudhury A (2001) The Arabidopsis AMP1 gene encodes a putative glutamate carboxypeptidase. Plant Cell 13:21152125 Henry Y, Vain P, DeBuyser J (1994) Genetic analysis of in vitro plant tissue culture responses and regeneration capacities. Euphytica 79:4558 Ho W, Vasil IK (1983) Somatic embryogenesis in sugarcane (Saccharum ofcinarum L.) I. The morphology and physiology of callus formation and the ontogeny of somatic embryos. Protoplasma 118:169180 Hou Y, Von Arnim AF, Deng X-W (1993) A new class of Arabidopsis constitutive photomorphogenic genes involved in regulating cotyledon development. Plant Cell 5:329339

repressed can promote a vegetative to embryogenic transition. However, the signalling pathway or mechanisms involved for acquisition of embryogenic competence are still incompletely known. Future work should be channelled more towards unravelling and understanding the signalling pathway involved in acquisition of embryogenic competence besides isolating for more molecular markers.

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