REVIEW

REGULATION OF IN VITRO WITH EMPHASIS ON THE ROLE OF ENDOGENOUS HORMONES

VÍCTOR M. JIMÉNEZ1 CIGRAS, Universidad de Costa Rica, 2060 San Pedro, Costa Rica.

ABSTRACT - Different aspects of in vitro somatic embryogenesis regulation are reviewed in this paper. General aspects, such as terminology, uses, stages of development and factors associated with the somatic embryogenesis , are described. Although a brief description of the effects of the addition of different plant growth regulators to the culture medium is given, the article is centered on the effect that endogenous hormone concentrations in the initial explants and in the tissue cultures derived from them could play in the induction and expression of somatic embryogenesis. It is significant that few systematic studies have been conducted, in which different species and hormone groups were compared in cultures with and without embryogenic capacity. Moreover, the lack of correlation between the results presented in different studies indicates that the hormone content of the cultures is not the only factor involved. ADDITIONAL INDEX TERMS: In vitro morphogenesis, regeneration, embryogenic competence, plant growth regulators, hormones. ABBREVIATIONS - 2,4-dichlorophenoxyacetic acid (2,4-D); abscisic acid (ABA); gibberellic acid 6 2 6 2 (GA3); indole-3-acetic acid (IAA); N -(D -isopentenyl) adenine (iP); N -(D -isopentenyl) adenosine (iPA); proembryogenic mass (PEM); zeatin (Z); zeatin riboside (ZR).

REGULAÇÃO DA EMBRIOGÊNESE SEMÁTICA IN VITRO COM ÊNFASE DO PAPEL DE HORMONIOS ENDÓGENOS

RESUMO - Neste trabalho se faz uma revisão de diversos aspectos da regulação da embriogêneses somático in vitro. Vários aspectos gerais a este fenômeno tem sido discutidos, tais como a definição de terminologia, descrição de eventuais aplicações, seus estados de desenvolvimento e outros fatores associados com sua indução e expressão. Embora se faça uma breve descrição do efeito da adição de diferentes reguladores de crescimento ao meio de cultivo, o artigo está centrado no efeito que as concentrações hormonais endogênas nos explantes iniciais e nos cultivos in vitro derivados deles podem ter na indução e expressão da embriogênese somática. Tem de se fazer ênfase na pouca quantidade de estudos sistemáticos realizados neste tema que comparem em várias espécies e diferentes grupos hormonais em cultivos com e sem competência embriogênica. Finalmente, indica-se que a falta de correlação entre os resultados destes poucos trabalhos parece indicar que os conteúdos hormonais endôgenos não são os únicos fatores envolvidos neste fenômeno. TERMOS ADICIONAIS PARA INDEXAÇÃO: Morfogêneses in vitro, regeneração de embriões, competência embrogênica, reguladores de crescimento, hormônios.

Received: 18/12/00 - Accepted: 11/06/01 1. CIGRAS, Univesidad de Costa Rica, 2060 San Pedro, Costa Rica. e-mail: [email protected] Regulation of in vitro somatic embryogenesis 197

INTRODUCTION (Umbelliferae). These early studies were very There are two alternative mechanisms by significant, because they confirmed Haberlandt’s which an explant can regenerate an entire plant, prediction that can arise from single cells in culture (i.e., cellular totipotency) (Kiyosue et al., namely organogenesis and somatic embryogenesis. Generally, in the first case, and form 1993; Höxtermann, 1997). The early history of sequentially and in response to appropriate culture somatic embryogenesis has been reviewed elsewhere (Raghavan, 1986; Halperin, 1995; conditions (mainly to the type and concentration of plant growth regulators present in the culture Krikorian & Simola, 1999). medium). This type of development is also Since these first studies, the number of higher plant species from which somatic embryos characterized by the presence of vascular connections between the mother tissue and the could be obtained and regenerated has continuously increased. This phenomenon has regenerating section (Terzi & Lo Schiavo, 1990). been documented in at least 200 and On the other hand, somatic embryogenesis can be described as the process by Angiosperm species (reviewed by Raemakers et al., which haploid or diploid somatic cells develop into 1995). Some species, however, are more recalcitrant than others regarding both initiation of structures that resemble zygotic embryos (i.e., bipolar structureswithout any vascular connection embryogenic cultures and regeneration of plants with the parental tissue) through an orderly series (Rao, 1996). Finding the right conditions to induce of characteristic embryological stages without somatic embryogenesis in different species and fusion of gametes (Williams & Maheswaran, 1986; cultivars is yet, for the greater part, based on trial Emons, 1994; Raemakers et al., 1995). One striking and error experiments (Jacobsen, 1991; Henry characteristic of the somatic embryo is its continuous growth resulting from the absence of et al., 1994): analyzing the effect of different culture conditions and media and modifying developmental arrest (Faure et al., 1998). Both especially the type and levels of plant growth processes, organogenesis and somatic embryogenesis, have been reported to occur in the regulators. However, the role that the genotype same explant (He et al., 1990), but originate from and its physiological condition play in this process has seldom been studied. This is a limitation for particular tissue layers or cells within explants (Osternack et al., 1999). improvement of long-term tissue cultures, to the A number of specialized examples of point that it restricted the development of somatic embryogenesis have been reported to fusion techniques and delayed for years occur in vivo, from both reproductive tissues such the development of genetic transformation as the nucellus and synergid cells, and somatic techniques beneficial for plant breeding of tissues such as somatic cells in () monocots (Henry et al., 1994). Only recently, by and leaf margins (Williams & Maheswaran, 1986; employing modern methods of hormonal Merkle et al., 1990, and also reviewed by Sharma & manipulations (transgenic and habituated ) in Thorpe, 1995). However, somatic embryogenesis is combination with precise and sensitive methods for nowadays best known as a pathway to induce hormone determination, has some progress been regeneration from in vitro tissue cultures. achieved in this area (Bangerth, 1993). Most of the Although it is widely accepted that the success achieved so far in understanding the first descriptions of in vitro somatic embryo mechanisms that govern the efficient regeneration production were carried out independently by of plants through somatic embryogenesis has been Steward et al. (1958) and Reinert (1959) working accomplished with model plant species and the with carrot, recently Krikorian & Simola (1999) transfer of these new technologies to major crop underlined the less noticed pioneer role of Harry species has been slow and difficult (Vasil, 1987). Waris, working with Oenanthe aquatica The successful induction of somatic embryos and

R. Bras. Fisiol. Veg., 13(2):196-223, 2001 198 Víctor M. Jiménez subsequent recovery of viable plants is not routine modeof culture permits easy scale-up transfers or efficient for the majority of species (Merkle with low labor inputs since embryos can be grown et al., 1995). The ability to understand the individually and freely floating in liquid medium; mechanisms involved in the induction and (c) unlike shoots, somatic embryos frequently expression of somatic embryogenesis in different originate from single cells and the embryogenic species will increase the number of genotypes cultures can be synchronized and purified so that capable of regeneration by this process. one can deal with practically pure cultures of homogeneous material; and, (d) plants derived APPLICATIONS OF SOMATIC from somatic embryos are less variable than those EMBRYOGENESIS derived by way of organogenesis (Ammirato, 1987; Somatic embryogenesis is a very Merkle et al., 1990; Terzi & Lo Schiavo, 1990; valuable tool for achieving a wide range of Osuga et al., 1999). The last point mentioned objectives, from basic biochemical, physiological above could be explained by an intolerance of and morphological studies, to the development of somatic embryos to mutations in any of the technologies with a high degree of practical numerous genes that must be necessary for a application. successful completion of ontogeny (Ozias-Akins & One of the main uses of somatic Vasil, 1988), while vegetative may be embryogenesis constitutes its employment as an more tolerant to mutations and epigenetic changes approach to investigate the initial events of zygotic (Merkle et al., 1990). embryogenesis in higher plants. Perhaps the Another application is in the production primary reason for the limited progress in of plants with different levels of ploidy; i.e., understanding the developmental events in plant obtaining haploid embryos by cultivating anthers embryos is that zygotic embryos of higher plants and raising triploids from have been consist of several tiny cells that grow within suggested and, to a very limited extent, exploited maternal tissues, such as the flowers or immature (Terzi & Lo Schiavo, 1990). Also, success in fruits, and it is quite difficult to collect sufficient inducing and the accomplishment of embryos for biochemical, physiological and long-term storage, together with the achievement morphological analyses of the biological events of encapsulation of somatic embryos, has opened that occur early in the developmental process. up the possibility for their use in the synthetic Somatic embryos provide a good model system by technology (Gray & Purohit , 1991; Gray et al., which such problems could be circumvented. The 1995; Litz & Gray, 1995). knowledge of many of the events that occur during The use of embryogenic callus and the early embryogenesis has resulted from suspension cultures, as well as somatic embryos experiments on somatic embryogenesis of a few themselves as a source of , has been plant species (de Jong et al., 1993; Kiyosue et al., exploited for a range of species, taking advantage 1993; Zimmerman, 1993). of the totipotency of these embryogenic cultures The mass propagation of plants through (Merkle et al., 1990). Embryogenic cultures have multiplication of embryogenic propagules is the proven to be especially valuable in providing a most commercially attractive application of source of regenerable protoplasts in graminaceous somatic embryogenesis (Merkle et al., 1990). species (Finch et al., 1991; Chang & Wong, 1994; Somatic embryogenesis has many advantages over Lyznik & Hodges, 1994; Funatsuki et al., 1996), organogenesis in this respect: (a) it permits the Citrus species (Jiménez , 1996), and forest trees culture of large numbers of ‘reproductive units’– (David, 1987; McCown & Russell, 1987). e.g., 60,000 to 1.35 million somatic embryos per Gene transfer into embryogenic plant liter of medium– with the presence of both cells is already challenging conventional plant and meristems in the same element; (b) the breeding, and has become an indispensable tool for

R. Bras. Fisiol. Veg., 13(2):196-223, 2001 Regulation of in vitro somatic embryogenesis 199 crop improvement. One of the most important The term ‘embryogenic cell’ is restricted prerequisites for genetic manipulation of plants to those cells that have completed their transition in vitro is the ability to grow somatic cells in sterile from a somatic (non-embryogenic) state to one in plant growth medium and to regenerate viable which no further exogenously applied stimuli, such plants from these cultures. Somatic as the application of growth regulators, are embryogenesis, therefore, is a more efficient necessary to produce the somatic embryo pathway for studies involving production of (Komamine et al., 1992; de Jong et al., 1993). The genetically transformed plants (Litz & Gray, 1995; cells that have reached this transitional state and Vicient & Martínez, 1998). have already started to become embryogenic, but Secondary or recurrent embryogenesis, that still require exogenously applied stimuli, are which is reported in at least 80 species (reviewed designated as competent cells (Mordhorst et al., by Raemakers et al., 1995), offers a great potential 1997). for in vitro production of embryo metabolites, such as lipids and seed storage proteins. However, since Induction of somatic embryogenesis the production costs are still higher than the extraction from natural , this technology is Induction of embryogenic growth in not yet commercially viable (Merkle et al., 1990). carrot cultures, as well as in cultures of many other Finally, the embryogenic development of species, appears to occurin one of two ways. somatic cells appears to be more sensitive to the Somatic embryos can form directly on the surface application of exogenous chemical compounds of an organized tissue such as a leaf or stem than the growth of whole plants or even callus segment, zygotic embryo, from protoplasts or from cultures. This offers the possibility of using microspores (i.e., the cells may be considered as in vitro screening and selection procedures to already determined for embryogenic development, identify plant genotypes resistant to certain factors, needing only permissive conditions to allow its such as aluminum toxicity or toxins produced by expression). They can also form indirectly via an pathogens (Merkle et al., 1990). intermediary step of callus or suspension culture (in these cases a more complex medium should be DEVELOPMENT OF SOMATIC used, including additional factors to induce EMBRYOGENESIS dedifferentiation and reinitiation of of already differentiated cells before they can express Somatic embryogenesis development has embryogenic competence) (Williams & Maheswaran, been divided into two main phases, namely, the 1986; Ammirato, 1987; Emons, 1994). one whereby differentiated somatic cells acquire Direct and indirect somatic embryogenic competence and proliferate as embryogenesis have also been considered as two embryogenic cells, and the phase whereby the extremes of a continuum (Williams & Maheswaran, embryogenic cells display their embryogenic 1986; Carman, 1990). Once induction of competence and differentiate into somatic embryogenic determined cells has been achieved, embryos. Both processes appear to be independent there appear to be no fundamental differences from each other and thus to be influenced by between indirect and direct somatic embryogenesis different factors. The former phase, called ‘Phase (Williams & Maheswaran, 1986). Emons (1994) 0’ by Komamine et al. (1992), ‘determination argued that in many systems in which phase’ by Rao (1996) and‘induction phase’ by embryogenesis has been designated as indirect, the Dodeman et al. (1997), has no direct counterpart in embryogenic callus is composed of young embryos zygotic embryogenesis (Emons, 1994). In this (pre-embryogenic masses or (pre-)globular work, the term ‘induction’ will be used to embryos) and that their further development designate the first phase and ‘expression’ for the dependson the duration of the application of the second one. inductive stimulus. If that period is relatively R. Bras. Fisiol. Veg., 13(2):196-223, 2001 200 Víctor M. Jiménez short, the process will be direct and if it is long, major groups of flowering plants, i.e., monocot then the process will be indirect. On the other embryos initiate only a single and hand, in other studies, direct embryogenesis has consequently do not proceed through a heart- beenused to describe the formation of an embryo shaped stage (Kaplan & Cooke, 1997). Yet another from a single cell without an intervening callus type of embryo development takes place in stage, although the embryo has arisen by means of conifers (Tautorus et al., 1991), which includes the dedifferentiation of a differentiated cell within three stages: globular, early cotyledonary and late the explant (c.f. references in Ammirato, 1987). cotyledonary embryos (Dong & Dunstan, 2000). In the carrot model described by In the aforementioned model of Komamine et al. (1992), competent single cells Komamine et al. (1992), describing the early (State 0) formed embryogenic cell clusters (State process of embryogenesis, ‘Phase 1’ is designated 1) in the presence of . These single cells are as the first phase in the expression of somatic considered as predetermined for embryogenesis. embryogenesis. This phase is induced by the During this phase, the cell clusters gained the transfer of State 1 cell clusters (already induced to ability to develop into embryos when auxin was express embryogenic development) to auxin-free removed from the medium, leading to the medium. During this phase, cell clusters development of State 1 cell clusters (Nomura & proliferate slowly and apparently without Komamine, 1985; Komamine et al., 1992). differentiation. After this phase, rapid cell division Embryogenic cells are unique: superficially they occurs in certain parts of cell clusters, leading to resemble meristematic cells, though they generally the formation of globular embryos (designated as are smaller, more isodiametric in shape, have ‘Phase 2’). In the following phase (Phase 3), larger, more densely staining nuclei and nucleoli, plantlets develop from globular embryos through and have a denser (Williams & heart- and torpedo-shaped embryos. Maheswaran, 1986; Carman, 1990). Expression of somatic embryogenesis might be triggered by different factors, depending Expression of somatic embryogenesis on species, cultivar, physiological conditions of the Once the inductionof an embryogenic donor plant and so on. However, as already state is complete, the mechanisms of pattern mentioned, the most common procedure is the formation that lead to the zygotic embryo are exclusion or reduction of the auxin (mainly common to all other forms of embryogenesis 2,4-dichlorophenoxyacetic acid [2,4-D]) (Mordhorst et al., 1997). Thus, somatic and zygotic concentration in the culture medium of embryos share similar gross ontogenies, with both embryogenic cultures induced with this plant typically passing through globular, heart-shaped growth regulator. and torpedo-shaped stages in dicots, or globular, scutellar (transition), and coleoptilar stages in FACTORS ASSOCIATED WITH monocots (Gray et al., 1995; Toonen & de Vries , EMBRYOGENIC COMPETENCE 1996). Schiavone & Cooke (1985) described an intermediate growth stage between globular and To take advantage of the previously heart-shaped embryos, that they termed the oblong mentioned potentialities that somatic embryo. Although heart- and torpedo-shaped embryogenesis offers, it is of great importance to embryos have traditionally been defined as understand the mechanisms underlying the separate stages of the embryo development, the transition from a somatic or a gametophytic cell to distinction between them is apparently based on an embryogenic cell and, in most cases, to be able the difference in size (Schiavone & Cooke, 1985). to regenerate plants from it (Mordhorst et al., 1997). These shape-based differences have their origin in At the same time, it is desirable to extend these the particular development of each of the two induction and expression capacities to those

R. Bras. Fisiol. Veg., 13(2):196-223, 2001 Regulation of in vitro somatic embryogenesis 201 species and cultivars that are recalcitrant to division does not form an embryo directly, but somatic embryogenesis, and are also of agronomic forms a proembryogenic mass (PEM), in which importance. When studying the factors involved in only one or a few cells can subsequently develop the control of somatic embryogenesis, it is very into an embryo (Komamine et al., 1992; Nuti Ronchi important to differentiate between those related to & Giorgetti, 1995). The rest of the PEM cells are the induction and those related to the expression of probably eliminated through a cycle of this event (Ermakov & Matveeva, 1994). Somatic programmed cell death, as observed in Norway embryogenesis induction can be spontaneous at spruce (Filonova et al., 2000). one end of the spectrum or may demand long and In a close relationship with in vitro complex treatments at the other end (Toonen & de embryogenesis, the orientation of the first cell Vries , 1996). Embryogenic competence is the term division has also been considered to be essential to used for describing the relative ease of inducing the establishment of the basic polarity of the somatic embryogenesis from tissue cultures zygotic embryo. However, a more extensive (Carman, 1990). survey of the embryological literature turned up a Explant (non-embryogenic) cells can be minimum of 19 different dicotyledonous families induced to an embryogenic state by a variety of in which the zygotes of at leastone species undergo procedures that usually include exposure to plant longitudinal, oblique, or even variable first growth regulators, pH shock, heat shock or divisions instead of the expected transverse treatment with various chemical substances. division (Kaplan & Cooke, 1997). However, it is still not clear whichchanges a In general, it can be concluded that a somatic cell must undergo in order to become an correct asymmetrical first division is not an embryogenic cell capable of forming an embryo. essential requirement for somatic or for zygotic There appears to be no single , universally embryogenesis. This does not imply that an applicable signal that renders cells embryogenic asymmetrical distribution of intracellular (Mordhorst et al., 1997). Moreover, in general only determinants is not important in the early stages of a very limited number of cells in any given explant , but rather that such a responds by becoming embryogenic (Toonen & de distribution is not necessarily visibly fixed by an Vries , 1996). asymmetrical first division. There are some other structural factors that could influence the capacity Structural factors forembryogenic induction in certain cells: The initiation of polarity in embryos is microtubule organization and cell wall (cell size often regarded as the first step in embryogenesis appears to play an important role in somatic (Warren Wilson & Warren Wilson, 1993). In carrot embryogenesis) (Toonen & de Vries, 1996). and Medicago it appears that the first division has McCabe et al. (1997) recently observed that a cell- to be asymmetric, producing two cells of different wall antigen on cells destined to form embryos sizes, in order to confer embryogenic competence segregates asymmetrically during a formative to the individual cells (Dudits et al., 1991). In the division, producing one daughter cell with a cell case of carrot, the first division of single wall antigen recognized by the antibody JIM8 and suspension cells capable of forming embryogenic the other without, and the epitope-free cells cells is unequal (Komamine et al., 1992), and only ultimately form somatic embryos. the smaller daughter cell will ultimately develop Another important point is the necessity into an embryo (de Jong et al., 1993). Komamine of the cells subjected to embryogenic induction, for et al. (1992), Tsukahara & Komamine (1997) and physical isolation from the surroundings. This is Sato-Nara & Fukuda (2000) reported a polarized the case with somatic embryos formed in DNA synthesis in these cells. In most species suspension cultures. This physical isolation leads showing embryogenic capacity, the asymmetric to varying degrees of physiological isolation R. Bras. Fisiol. Veg., 13(2):196-223, 2001 202 Víctor M. Jiménez caused by loss of plasmodesmata between et al., 1991, 1992). The addition of surrounding cells, interrupting symplastic arabinogalactan proteins, isolated from the culture continuity and reducing electrical coupling (Warren medium of embryogenic carrot lines and from dry Wilson & Warren Wilson, 1993). carrot seeds, was capable of promoting the formation of PEMs, even in previously non- Physiological factors embryogenic carrot cell lines. Similar proteins, but Although plant growth regulators play a isolated from the medium of non-embryogenic key role in inducing somatic embryogenesis, there lines, acted negatively on the formation of PEMs are many other factors that have been found which (de Jong et al., 1993). McCabe et al. (1997) found could affectthe disposition of a particular tissue to that cells competent to become embryogenic undergo somatic embryogenesis. The range of require a soluble signal from other cells to trigger possible induction treatments suggests that it is somatic embryogenesis, and postulated that signal unlikely that a single inducing molecule is is an . The way in which responsible (Toonen & de Vries, 1996). secreted proteins influence somatic embryogenesis Examples of these other factors that can is not known, but it is reasonable to postulate that direct the transition from somatic cells to cells able their function can be explained in terms of an to form embryo-like structures are: in Citrus effect on particular cell wall polymers (van Holst suspension cultures a change in carbon source et al., 1981). For a review of the role of these from sucrose to glycerol (Ben-Hayyim & Neumann, proteins in this process, refer to de Jong et al. 1983; Gavish et al., 1991; Jiménez & Guevara, (1993), Kiyosue et al. (1993) and Schmidt et al. + 1996); in carrot the NH4 concentration (Smith & (1994). Krikorian, 1989) and pH changes (Smith & Meijer et al. (1999) observed that co- Krikorian, 1990, 1992) in the culture medium; in culture of Arabidopsis thaliana suspension culture Araujia sericifera the light quality (Torné et al., aggregates interferes with the development of 2001), in Brassica microspores a temperature carrot somatic embryos beyond the globular stage. shock (Pechan & Keller, 1988); also pre-treatment These authors attribute this effect to the release of of donor plants and subculture duration (Mórocz previously accumulated 2,4-D by the Arabidopsis et al., 1990), to name only a few factors. A more cultures. Moreover, Kobayashi et al. (2000) found detailed description of some of these factors was that somatic embryogenesis is strongly inhibited in given by Tisserat et al. (1979), Sharp et al. (1980), cultures of carrot cells when the cell density is Tulecke (1987) and Harada (1999). high, and successfully isolated and identif ied the Culture density of cell suspensions could inhibitory factor 4-hydroxybenzyl alcohol, which be another important factor that affects somatic was found to strongly inhibit the formation of embryogenesis. While a high cell density somatic embryos when added to the culture (105 cells/ml) is required for the formation of medium at a concentration equal to that found in embryogenic cell clusters from single cells high-cell-density cultures. Differences in the (Nomura & Komamine, 1985), a lower cell density contents of reducing sugars and starch have been (2 x 104 cell/ml) favors the development of reported to be characteristic of embryogenic and embryos from embryogenic cells (Fujimura & non-embryogenic calli from Medicago arborea, Komamine, 1979). This may be related to the with higher sugar concentrations and lower starch secretion of proteins and/or other cellular factors content in the embryogenic cultures than in the into the culture medium. In fact, secreted non-embryogenic cultures (Martin et al., 2000). (extracellular) and constitutive (intracellular) Gene expression proteins are also considered to play a very important part in induction of somatic and differentiation are embryogenesis (de Vries et al., 1988a,b; Gavish regulated directly or indirectly by changes of gene R. Bras. Fisiol. Veg., 13(2):196-223, 2001 Regulation of in vitro somatic embryogenesis 203 expression, especially during embryogenesis been divided. On the other hand, some mutations (Dong & Dunstan, 2000). Molecular studies on of Arabidopsis thaliana have been used to plant embryogenesis began in the 1980’s, and since characterize the induction phase, i.e., the first stage then they have evolved in an interesting way. during somatic embryogenesis. Initial studies of zygotic embryogenesis were Most genes expressed differentially generally designed to estimate the number of during somatic embryogenesis belong to the late different RNAs present in developing seeds, to embryo-abundant (lea) genes. Proposed functions examine the spatial and temporal distribution of for the products of this family of genes arethe distinct RNA species, to isolate and to characterize protection of cellular structures in mature embryos the genes that code for abundant seed proteins, during seed desiccation and prevention of particularly the seed-storage proteins, and to precocious of the zygotic embryos identify the cis-acting regulatory sequences and during seed development (reviewed by Wilde trans-acting DNA-binding proteins that regulate et al., 1995 and Dong & Dunstan, 2000). Several expression of seed-specific genes (Meinke, 1995). genes expressed in carrot somatic embryos code However, the basic experimental strategy for secreted extracellular proteins. One gene for molecular analysis of somatic embryogenesis product (EP1), with homology to Brassica S-locus has mostly relied on comparing genes and proteins glycoproteins, is present in nonembryogenic callus being expressed in embryogenic and non- but not in somatic embryos themselves. Another embryogenic cells as well as in the different stages gene that produces a lipid transfer protein (EP2) of embryogenesis (Rao, 1996). Gene expression has been particularly useful as a marker for studies during the different stages of this process epidermal cell differentiation during have been carried out (reviewed by Henry et al., embryogenesis. The precise role of these 1994; Kawahara and Komamine, 1995; Meinke, extracellular proteins remains to be established, but 1995; Wilde et al., 1995 and Dong & Dunstan, they may be involved in the regulation of cell 2000) and suggest that the number of genes expansion and the maintenance of biophysical specifically expressed during these events is rather features required for morphogenesis. Perhaps the limited (Komamine et al., 1992; Ermakov & most unexpected finding involves a secreted Matveeva, 1994; Dodeman & Ducreux, 1996; glycoprotein (EP3) that rescues a temperature- Schrader et al., 1997), and that changes in protein sensitive mutant of carrot (ts11 ) that fails to patterns are highly regulated posttranscriptionally, complete the transition from a globular to heart at the mRNA level (Komamine et al., 1992; Wilde stage of somatic embryogenesis (Meinke, 1995; et al., 1995). Additionally, Dodeman & Ducreux Sugiyama, 2000). Another group of genes that are (1996) indicate that changes in hormonal levels in developmentally regulated in carrot suspension tissue cultures may modify the synthesis of some cultures is constituted by the Dc3, Dc8, J4e and somatic embryogenesis-specific-proteins. ECP31 genes. They are expressed at different Recently, Kitamiya et al. (2000) succeeded in momentsduring embryo development and localized isolating two genes that were induced after in different cell groups within the PEMs and exposure of carrot hypocotyls to high embryos (Wilde et al., 1995). Dudits et al. (1995) concentrations of 2,4-D for 2 hours, a treatment indicate that the gene expression is expected to be that initiated somatic embryogenesis directly on different during the processes of embryogenic these explants. commitment in primary explants or fully Cell cultures of carrot that are growing as differentiated somatic cells from those that act in unorganized callus in the presence of 2,4-D have suspension cultures with proembryogenic been used to study genetics during expression of structures, such as in the case of carrot. somatic embryogenesis, the latter of the two phases In recent years, there has been a shift in which, as previously described, this process has toward combining the tools of molecular

R. Bras. Fisiol. Veg., 13(2):196-223, 2001 204 Víctor M. Jiménez with the power of genetics. Future research in biochemists, physiologists, and cell biologists, plant molecular biology must focus not simply on many of whom could use mutants defective in isolating and characterizing large numbers of genes basic cellular processes. Embryonic mutants also expressed during plant embryo development, but differ in the initial site of gene action. The primary also on determining the biological significance of defect in some embryonic mutants is limited to the these genes by demonstrating what happens when endosperm tissue. Altered development of the their function is disrupted. This can be embryo in these mutants is often an indirect accomplished either by creating transgenic plants consequence of endosperm failure. The primary that express an antisense construct or by working defect in other mutants appears to be restricted to with genes that have already been disrupted the embryo proper. Although the most extensive through loss-of-function mutations (Meinke, studies of embryonic mutants have dealt with 1995). maize and A. thaliana, related mutants have also The general strategy in genetic analysis is been described in barley, carrot, rice, and peas. to use mutants either as markers of cell lineages or Most of these recessive loss-of-function mutations as vehicles for the identification of essential genes. were induced with chemical mutagens, X rays, Genetic analysis has played an increasingly transposable elements, or T-DNA from important role in recent studies of plant Agrobacterium tumefaciens (Meinke, 1995). development. However, not all genes can be identified readily by recessive loss-of-function Molecular markers mutations. Genes that are duplicated in the The search for markers of plant genome, genes that are required for early stages of embryogenesis is an important aspect of modern gametogenesis, and genes that perform functions plant breeding (Schel et al., 1994). Several that are redundant, nonessential, or detectable only physiological, biochemical and molecular markers in unique circumstances often escape detection in associated with embryogenic competence of cells mutant screens. The power of genetics is not that have been reported, including isozymes and it leads to the identification of every transcriptional molecular markers, to be discussed in the unit within the genome, but rather that it allows following paragraphs, and plant hormones, which one to focus on genes that must be expressed in will be discussed later. order for growth and development to proceed in a Isozyme patterns are helpful tools for a normal manner (Meinke, 1995). better understanding of the basic mechanisms of This strategy has been used more readily cellular differentiation and further plant to try to understand the events related to zygotic development. Apart from the classical studies in embryogenesis. Several different classes of which isozymes were used as markers for events at embryonic mutantshave been identified in higher the later stages of the growing plant (e.g., plants. Many mutants are defective in early stages organogenesis or seed germination), during the last of cell division and morphogenesis. Others fail to twenty years their usefulness as markers during accumulate pigments and storage materials during early development of the plant (i.e., early embryonic maturation. Still others are disrupted in embryogenesis) was also demonstrated.Tissue the preparation for dormancy and germination. culture techniques and, more specifically, somatic Many of these mutants are likely to be defective in embryogenesis allowed the application of isozyme genes with housekeeping functions that first analysis to the embryogenic process, because they become essential during embryo development. permitted the availability of relatively high Although this information may be discouraging to amounts of plant material in the desired developmental biologists interestedin finding genes developmental stage. that play a direct role in the regulation of plant Coppens & Dewitte (1990) found the embryogenesis, it should be very useful for esterase system to be very sensitive for detection of

R. Bras. Fisiol. Veg., 13(2):196-223, 2001 Regulation of in vitro somatic embryogenesis 205 embryogenesis in barley callus before somatic (6%), Picloram (5%) and Dicamba (5%). In the embryos are formed. In carrot, two esterase case of , N6-benzylaminopurine was isozyme systems are differentially expressed in used most often (57%), followed by kinetin (37%), embryogenic and non-embryogenic cells (Chibbar zeatin (Z) (3%) and thidiazuron (3%). et al., 1988). Tchorbadjieva & Odjakova (2001) In those cases in which the exogenous recently found an acidic-esterase isozyme (36 kDa application of has proved to be the most and an isoelectric point of 3.8) present in efficient treatment to induce somatic embryogenic suspension cultures of Dactylis embryogenesis, further development of the existing glomerata but not in non-embryogenic cultures. somatic embryos has been achieved by reducing or Also, it has been observed that the pattern of acid removing auxin fromthe culture media. Although phosphatase showed qualitative differences the process of embryo induction from cells in between embryogenic and non-embryogenic coffee culture is not fully understood, it is now generally callus cultures (Menéndez-Yuffá & García, 1996). believed that, in the continued presence of auxin, a An extensive review on the use of isozymes as differential change in gene expression (probably biochemical markers in somatic embryogenesis has associated with increased demethylation of DNA; been already published (Schel et al., 1994). Lo Schiavo et al., 1989) in PEMs occurs (Litz & There are several candidate genes that Gray, 1995). Under these circumstances, the PEMs could be used as molecular markers of single within the culture synthesize all the gene products competent cells (Schmidt et al., 1997). One of necessary to complete the globular stage of these genes, the Somatic Embryogenesis Receptor- embryogenesis. At that point, the PEMsalso like Kinase (SERK) gene, was found to mark contain many other mRNAs and proteins whose single Daucus and Dactylis suspension cells that continued presence generally inhibits the are competent to form somatic embryos (Schmidt continuation of the embryogenic program. The et al., 1997; Somleva et al., 2000). removal of auxin results in the inactivation of a number of genes so that the embryogenic program EFFECT OF EXOGENOUSLY APPLIED can now proceed. The observation that some PLANT GROWTH REGULATORS ON carrot cell lines are able to develop to the globular SOMATIC EMBRYOGENESIS stage, but not beyond in the continued presence of Although auxins, which are known to auxin, suggests that new gene products are needed mediate the transition from somatic to for the transition to the heart stage and that these embryogenic cells, are the agents generally used to new products are synthesized only when induce embryogenesis, the effect of other plant exogenous auxin is removed (Zimmerman, 1993). growth regulators on this phenomenon must not be However, as stated earlier in this work, the number overlooked. While in Angiosperm monocots, of genes directly involved in development of primary embryogenesis was exclusively induced somatic embryos seems to be rather limited. by auxin-supplemented media, there is a large The effect of the addition of other plant variation of growth regulators used to induce growth regulators is not so well documented. It somatic embryogenesis in dicot species. From a has been observed that the addition of abscisic acid list of 65 dicot species reviewed by Raemakers (ABA) inhibits the precocious germination of the et al. (1995), somatic embryogenesis was induced somatic embryos and allows them to mature into in 17 species on hormone-free media, in 29 species ‘normal-shaped’ plants, as observed in grapevine on auxin-containing media and in 25 species on (Rajasekaran et al., 1982; Goebel-Tourand et al., -supplemented media. Among auxins, 1993). Bell et al. (1993) found that explants of the most frequently used was 2,4-D (49%) embryogenic genotypes of Dactylis glomerata followed by naphthalene acetic acid (27%), indole- responded to the inclusion of ABA in the culture 3-acetic acid (IAA) (6%), indole-3-butyric acid medium by increasing the number of somatic

R. Bras. Fisiol. Veg., 13(2):196-223, 2001 206 Víctor M. Jiménez embryos formed. Nishiwaki et al. (2000) observed main difference between genotypes with various that of carrot formed somatic embryos grades of competence (Bhaskaran & Smith, 1990). when cultured on medium containing ABA, with Fot this reason the endogenous hormone the number of embryos originated per explant levels and their relation to the embryogenic dependent on the ABA concentration employed. competence of the explants may be the key to Despite the wide range of physiological effects of induction and expression of somatic gibberellins, their effect when added to culture embryogenesis in recalcitrant genotypes through media, primarily as gibberellic acid (GA3), has amendments to the culture medium, with been minimal (Krikorian, 1995). Exogenous substances that may mimic the inductive condition application of GA3 has been reported to inhibit (supplying a deficiency or counteracting an somatic embryogenesis and somatic embryo excess), to develop or optimize in vitro protocols development in several species; but it was also for somatic embryo production, maturation, and reported that this substance is required for conversion to plantlets (Merkle et al., 1995). At the germination of the mature somatic embryos if same time, the characterization of the differences chilling is not applied (Takeno et al., 1983 and between embryogenic and non-embryogenic cells references therein). Even in those cases in which is necessary if the mechanisms involved in the the addition of cytokinins as the sole plant induction and maintenance of embryogenic regulator is effective in inducing somatic competence of somatic cells are to be elucidated embryogenesis ,, the stimulatory effect of these (Kiyosue et al., 1993). substances is not universal, and their addition As previously stated in this work, in should often be coupled with that of auxins to most early studies on hormonal regulation of obtain the desired effect (reviewed by Merkle et al., physiological processes in plants, especially in 1995). in vitro cultures, more attention was paid to the effects of the addition of plant growth regulators Effect of endogenous hormones on somatic than to the possible role of endogenous embryogenesis hormonal levels in the tissues. Very little is It has been frequently observed that known about the possible interactions of an embryogenic competent and incompetent callus endogenous phytohormone system with the sections are produced in the same explant, exogenous growth regulators supplied to the indicating that even genetically identical cells nutrient medium. It seems probable that the respond differently to a particular stimulus, with a observed responses of cell culture systems, after minority of the cells being responsive. Several a growth regulator supplement, are related to changes should occur for reprogramming a cell to such interactions (Neumann, 1988; Carman, an embryogenic competent state, the first one 1990). Evidence for this has been reported by being the termination of the current gene Liu et al. (1998), who observed an accumulation expression patterns, permitting their replacement of endogenous IAA in soybean hypocotyl with an embryogenic program, which does not explants after their treatment with occur in all cells at the same time, and in some napththaleneacetic acid and indole-3-butyric acid cells does not occur at all (Merkle et al., 1995). (two exogenously applied auxins). Anatomical and physiological differences between There are some reports that relate the embryogenic and non-embryogenic cultures are endogenous hormone levels in the initial explants expressed when such changes occur. Among these (Carnes & Wright, 1988; Kopertekh & Butenko, differences, the endogenous hormone levels should 1995; Hess & Carman, 1998) and in the callus or be of great importance, since they regulate the cell suspension cultures derived from them (Epstein processes of explant differentiation in culture et al., 1977; Fujimura & Komamine, 1979; (Grieb et al., 1997) and are postulated to be the Rajasekaran et al., 1982; Takeno et al., 1983; Li & R. Bras. Fisiol. Veg., 13(2):196-223, 2001 Regulation of in vitro somatic embryogenesis 207

Neumann, 1985; Rajasekaran et al., 1987b; Kiyosue related this finding to a reduction in the precocious et al., 1992; Michalczuk et al., 1992a; Sasaki et al., germination rate. Precocious germinationand 1994; Kopertekh & Butenko, 1995; Hess & Carman, callus formation have been frequently reported as 1998) to the morphogenetic competence of a alternative responses of immature embryos to particular genotype. However, most of these in vitro culture (i.e. the occurrence of one studies were limited to only one or a few diminishes the chances of the other to occur) hormones, and to one plant species. Usually, (Carman, 1988; Qureshi et al., 1989). Thus, a information presented in different articles can not reduction in precocious germination would favor, be put together and used to obtain a general view indirectly, callus formation. Working with maize, of the role of endogenous hormone levels in Jiménez & Bangerth (2001c) did not find any somatic embryogenesis, because distinct genotypes difference in the levels of five hormones analyzed were used and also because the analytic methods between two genotypes differing in their degree of were not the same in the different studies. It is competence to conduct somatic embryogenesis. very important to compare different plant species In similar studiesanalyzing the whole simultaneously, using the same analytic al grains of wheat instead of isolated zygotic procedure, because from the study of a single embryos, Hess & Carman (1998), working with wheat, found lower levels of IAA, ABA, N6-(D2- organism, it is impossible to determine whether the 6 2 particular features are unique to that organism or isopentenyl) adenine (iP) and N -(D -isopentenyl) generally applicable to other species (Kaplan & adenosine (iPA) and Carnes & Wright (1988) Cooke, 1997). found higher levels of total IAA and lower levels of Z and zeatin riboside (ZR) in the most Endogenous hormone levels in the initial competent of two maize varieties. However, it has explants been observed that the whole kernel hormone level poorly reflects the levels in the immature embryos There are contrasting reports in relation since the endosperm constitutes the majority of to the effect of the endogenous hormone levels in kernel dry matter, and, as was demonstrated by the initial explants on determining the ability of a Jiménez & Bangerth (2001b, 2001c), hormone particular genotype to conduct somatic levels in the endosperm might vary greatly in embryogenesis. Whereas differences in hormone relation to those of the immature embryos. levels among competent and non-competent plant In three other monocot systems, genotypes have been documented, other Pennisetum purpureum (Rajasekaran et al., 1987a), investigations reported no discrepancy, or even that Dactylis glomerata (Wenck et al., 1988) and the differences found did not account for variations Medicago falcata (Ivanova et al., 1994), in which in the degree of competence. In many cereal species, immature zygotic leaf sections were used as the initial explants, embryos are the primary source of explants higher IAA levels were found in embryogenic than employed to establish embryogenic cultures. in non-embryogenic explants. Ivanova et al. (1994) When analyzing wheat immature embryos of two also found a positive correlation between the cultivars differing in their degree of competence, ability to respond to a 2,4-D induction treatment Kopertekh & Butenko (1995) found higher IAA and and endogenous IAA levels. The line with the ABA and lower cytokinin levels in the most shortest induction phase had the highest IAA level. competent genotype. However, in a similar The other embryogenic line needed an induction experiment, but using another set of wheat period 4 to 5 times longer and had an IAA content genotypes also differing in their embryogenic 4 times lower. In the same study, a negative competence, Jiménez & Bangerth (2001b) found relation between the endogenous ABA level and higher ABA levels in the competent one as the the embryogenic potential was observed. unique difference among them. The latter authors However, in Pennisetum purpureum leaves, higher

R. Bras. Fisiol. Veg., 13(2):196-223, 2001 208 Víctor M. Jiménez levels of ABA (3- to 4-fold) were found in the cytokinins in of three Corylus avellana more embryogenic sections than in the less genotypes with different levels of embryogenic embryogenic (Rajasekaran et al., 1987b). The same capacity; however, they showed a very different iP- group found that treatments which lowered ABA type/Z-type cytokinin ratio. They reported higher levels also inhibited somatic embryogenesis, and endogenous levels of iP and lower of Z, correlated that this inhibition could be overcome by with higher levels of embryogenic competence, exogenous ABA in the medium (Rajasekaran et al., when compared to those of less competent 1987a). genotypes. There are also contrasting reports in The contrasting results cited above seem relation to the role that endogenous gibberellin to indicate that differences in the endogenous levels in the initial explants could play in hormone levels in the initial explants of cereals are determining the embryogenic competence. As not indicative of their embryogenic competence. previously mentioned, Jiménez & Bangerth Supporting this conclusion, Jiménez & Bangerth (2001b, 2001c) did not find any difference in the (2001d) reported marked differences in the IAA endogenous gibberellins levels (GA1,3,20) between and gibberellins levels in immature zygotic barley embryogenic and non-embryogenic genotypes of embryos, differences that did not affect their wheat and maize. In another study (Jiménez & degree of competence. Bangerth, 2001d), the same authors indicate that differences in the endogenous levels of the same Endogenous hormone levels in embryogenic and substances in two genotypes of barley did not non-embryogenic cultures condition the response to the culture conditions Compared to the few studies available on evaluated. Also, Rajasekaran et al. (1987a) the effect of the hormone status of the initial observed that neither paclobutrazol nor the reduced explant on embryogenic competence, more levels of gibberellins, which may have resulted research has been carried out in relation to the from its application, altered the embryogenic endogenous hormone levels in embryogenic and character of the explants. In contrast, Hutchinson non-embryogenic callus cultures. Several reports et al. (1997) reported that both exogenously indicate that higher endogenous free IAA levels supplied as well as endogenous gibberellins play a are characteristic of embryogenic ally competent negative role in the induction of somatic callus lines, as observed in carrot (Li & Neumann, embryogenesis in geranium (Pelargonium x 1985; Sasaki et al., 1994, Jiménez & Bangerth, hortorum) hypocotyl explants. 2001a), Pennisetum purpureum (Rajasekaran et al., In contrast to the results obtained in 1987b), sugarcane (Guiderdoni et al., 1995), wheat alfalfa (Ivanova et al., 1994), wheat (Jiménez & (Jiménez & Bangerth, 2001b) and maize (Jiménez Bangerth, 2001b) and maize (Jiménez & Bangerth, & Bangerth, 2001c). On the other hand, 2001c), which indicate that the endogenous Michalczuk et al. (1992a) did not find differences in cytokinin levels in the initial explants do not free and conjugated IAA between embryogenic apparently determine their embryogenic and non-embryogenic carrot cultures, and Besse competence, Wenck et al. (1988) reported that low et al. (1992) did not observe great differences in endogenous levels of cytokinins were the IAA content of callus lines of Elaeis guineensis characteristic of leaves of Dactylis glomerata with that differ in their embryogenic response. high embryogenic capacity. Supporting the latter Rajasekaran et al. (1987a) found that low finding, Rajasekaran et al. (1987a) reported that auxin levels coincided with high cytokinin levels both non-embryogenic leaf regions and callus of in non-embryogenic callus cultures of Pennisetum purpureum contained higher levels of P. purpureum and postulated that the lower levels cytokinins than the highly embryogenic material. of IAA in non-embryogenic callus may be a Centeno et al. (1997) found similar contents of total consequence of the uptake and accumulation of

R. Bras. Fisiol. Veg., 13(2):196-223, 2001 Regulation of in vitro somatic embryogenesis 209 cytokinins, which, at optimal concentrations, have found higher gibberellin levels in embryogenic been shown to stimulate the formation of maize lines and, in contrast, Noma et al. (1982) isoperoxidase and IAA oxidase, enzymes related high levels of polar gibberellins (probably responsible for irreversible degradation of IAA GA1) to an absence of embryogenic competence in (Lee, 1971). However, Jiménez & Bangerth (2001a, carrot. 2001b, 2001c) could not confirm this hypothesis Concerning the endogenous levels of since they did not find any coincidence between cytokinins, Ernst et al. (1984) and Ernst & low levels of free IAA and high levels of Oesterhelt (1984, 1985), working with anise, cytokinins in embryogenic and non-embryogenic concluded that the cytokinin levels in the callus callus cultures of carrot, wheat and maize. cultures seemed to be more related to the growth A reduction in the embryogenic capacity rate of the calli than to their embryogenic of the explants with time in culture under competence. Similar conclusions were postulated inductive conditions (specially on medium by Jiménez & Bangerth (2000) in grapevine. containing 2,4-D) has been reported by Smith & However, Rajasekaran et al. (1987a) found levels of Street (1974) and Filippini et al. (1992). Rajasekaran cytokinins in non-embryogenic callus at least two et al. (1987b) in P. purpureum, Kopertekh & times higher than in embryogenic callus, after 10 Butenko (1995) and Jiménez & Bangerth (2001b) in days of culture of Pennisetum purpureum leaf wheat and Jiménez & Bangerth (2001c) in maize explants. Guiderdoni et al. (1995) reported higher found that decreased embryogenic competence was levels of iP and iPA in embryogenic calli than in characterized by a reduction in the endogenous the non-embryogenic calli of sugarcane, the free IAA, practically to the levels present in the opposite for Z, andno differences in the ZR levels. non-embryogenic lines. Higher ABA levels in embryogenic Habituated cultures and endogenous hormones callus lines, when compared to non-embryogenic Habituated cultures are those that can lines, were reported by Rajasekaran et al. (1987b) in proliferate in a culture medium without the Pennisetum purpureum, Kiyosue et al. (1992) and supplement of exogenous plant growth regulators Jiménez & Bangerth (2001a) in carrot, Guiderdoni (Meins, 1989). There is some controversy in et al. (1995) in sugarcane and Jiménez & Bangerth relation to the hormone physiology of habituated (2000) in grapevine. Kiyosue et al. (1992) and non-habituated cells. While similar postulated that high endogenous ABA levels could concentrations of hormones in both habituated and be necessary to induce or maintain somatic non-habituated cell types have been reported embryogenesis in carrot. Contrary to these results, (Kevers et al., 1981), higher hormone Etienne et al. (1993) found that high levels of ABA concentrations were also found in habituated than accumulated in non-embryogenic calli of Hevea in non-habituated cells (du Plessis et al., 1996 and brasiliensis, but remained at a low level in the references therein). It has been argued that embryogenic callus. These latter authors habituation may be due to the elevated levels of postulated that high ABA levels are incompatible cytokinins in the habituated cells (du Plessis et al., with the initiation and then the development of 1996). However, Das and Saha (1995), analyzing somatic embryos. the endogenous cytokinin levels in habituated and No defined pattern was observed in the non-habituated nucellar callus of sweet orange, endogenous levels of gibberellins when comparing found higher levels of Z, ZR and Z glucoside in the embryogenic with non-embryogenic callus second callus type. cultures. While Jiménez & Bangerth (2001a) in It was previously mentioned that carrot, Jiménez & Bangerth (2001b) in wheat and embryogenic callus cultures developed on culture Jiménez & Bangerth (2000) in grapevine did not medium containing 2,4-D are characterized by find any difference, Jiménez & Bangerth (2001c) higher endogenous IAA contents than those of the

R. Bras. Fisiol. Veg., 13(2):196-223, 2001 210 Víctor M. Jiménez non-embryogenic cultures. Since Michalczuk et al. embryogenesis. It has been reported that the (1992b) postulated that the 2,4-D in the culture culture of explants in 2,4-D-containing medium, medium is the cause of this increase in the the classic induction treatment for many species, endogenous IAA levels, it is conceivable to think increases the endogenous auxin levels in the that in the habituated cultures, grown on a explants (Michalczuk et al., 1992b). hormone free medium, this increase does not It has also been observed that polar occur. In grapevine, Jiménez & Bangerth (2000) transport of auxin is essential for the establishment reported that competent callus cultures did not of bilateral symmetry during embryogenesis in show this characteristic pattern of higher IAA dicotyledonous somatic (Schiavone & Cooke, 1987) levels, instead the levels were similar in and zygotic (Liu et al., 1993) embryos, and more embryogenic and non-embryogenic cultures. recently it was also demonstrated for Working with habituated Citrus callus and cell monocotyledonous zygotic embryos (Fischer & suspension cultures, Jiménez et al. (2001) found Neuhaus, 1996). For this gradient to be established, that the treatment that stimulated the further relatively high levels of free IAA may be necessary development of the somatic embryos also in the competent tissues. However, once the stimulated auxin and cytokinin accumulation. stimulus for the further development of the somatic However, these authors did not make comparisons embryos is given (i.e., through reduction or between embryogenic and non-embryogenic removal of 2,4-D from the culture medium), those cultures. levels must be reduced, to allow the establishment Limanton-Grevet et al. (2000) analyzed of the polar auxin gradient. If the levels are the endogenous levels of IAA, ABA, Z, ZR, iP and extremely low or high, or if they do not diminish iPA in eight embryogenic habituated callus lines after the induction treatment, the gradient cannot derived from six different Asparagus officinalis be formed, and thus somatic embryogenesis cannot genotypes, maintained on hormone-free medium be expressed. Indoleacetylaspartate as been found for more than one year. Even when a great in high levels in embryogenic callus of ‘Shamouti’ variaton in the intensity of the secondary orange (Epstein et al., 1977), and it was suggested embryogenesis was observed between lines, this that through this mode of IAA conjugation, auxin could not be related to the hormone metabolism, levels in embryogenic callus are reduced to a level since no significant differences were found induc ive to active embryogenesis. between distinct embryogenic lines. Endogenous levels of ABA also appear PROPOSED ROLE FOR EACH HORMONE to be significant in some monocots for initiation of GROUP DURING SOMATIC embryogenic cultures (Bhaskaran & Smith, 1990). EMBRYOGENESIS Its role as an inhibitor of precocious germination when immature zygotic embryos are used as initial Although the endogenous hormone levels explants, thus favoring indirectly callus formation, by themselves do not account for all the was discussed by Jiménez & Bangerth (2001b). phenomena observed during induction and The role of ABA in somatic embryogenesis may be development of somatic embryogenesis, a general exerted through regulation of certain genes (e.g., view of the importance of each hormone group can DC8) that are thought to be involved in desiccation be postulated. and maturation phases of embryogenesis Auxin is considered to be the most (Hatzopoulos et al., 1990). Rajasekaran et al. important hormone in regulating somatic (1987a,b) proposed that ABA could exert its role embryogenesis (Cooke et al., 1993). Both the on somatic embryogenesis by regulating endogenous contents and the application of carbohydrate metabolism, via inhibition of a- exogenous auxins are determin ing factors during amylase activity. There are several instances the induction and expression phases of somatic where applied ABA has been shown to stimulate

R. Bras. Fisiol. Veg., 13(2):196-223, 2001 Regulation of in vitro somatic embryogenesis 211 cell division and DNA synthesis, callus production concentration and the response of the explants was and shoot morphogenesis, and to inhibit peroxidase observed, led Trewavas (1981) to postulate that the activity, an enzyme that, as previously mentioned, sensitivity of the tissues to a change in the causes an irreversible degradation of IAA hormone concentration is more important than the (Rajasekaran et al., 1987a and references cited change in the concentration itself. This proposal therein). Senger et al. (2001), using a homozygous generated a lot of controversy at that time, but it transgenic line of Nicotiana was supported by several observations, such as the plumbaginifoliaconstitutively expresing an anti- fact that immature flowers and fruits did not ABA single chain fragment variable antibody in respond to ethylene application, but the mature the endoplasmic reticulum, clearly demonstrated ones did (Davies, 1995). that ABA is important for the formation of pre- Several reports, such as those in which globular structures. no differences between the endogenous levels of The role of exogenous and endogenous most hormones evaluated in the initial explants of gibberellins in somatic embryogenesis has not been competent and non-competent genotypes were studied in detail. The results of different reports found (Jiménez &Bangerth, 2001b, 2001c) or that seem to indicate a minor role for endogenous in which differences in the hormone levels did not gibberellin levels, both in the initial explants and seem to play any role in determining the the callus cultures (Jiménez & Bangerth, morphogenetic capacity of the explants (Jiménez & 2001a,b,c,d). Bangerth, 2001d), demonstrate that there are, There is support for the concept that certainly, other factors, besides the endogenous cytokinins , in general, are important during the levels of plant hormones, that determine the initial cell division phase of somatic competence of the tissues to develop embryogenic embryogenesis, but not for the later stages of cells. This kind of result seems to support the embryo development and maturation in carrot postulate of Trewavas; however, now it is widely (Fujimura & Komamine, 1980), anise (Ernst et al., accepted that both the sensitivity of the tissues to 1984; Ernst &Oesterhelt, 1985) and orchardgrass the plant hormones and the concentration of the (Wenck et al., 1988). This suggests that cytokinins hormones, are important and modulate the may have a role in cell division, but not in embryo responses observed in most cases. Sensitivity has differentiation (Danin et al., 1993). When been shown to contribute in a higher degree to, at analyzing the individual role of the iP- and Z-type least, gravitropic responses, cytokinins, Centeno et al. (1997) postulated that the regeneration, cambial growth, seed dormancy, role the first group in the embryogenic response germination, cell division, cell extension, ripening, might be as biosynthetic precursors to supply the senescence, abscission, stomatal aperture, large amount of active cytokinins required for the flowering and amylase formation (reviewed by stimulation of cell division prior to somatic embryogenesis. In the general proposed pathway Trewavas , 1991). There is some evidence that sensitivity of cytokinin biosynthesis in prokaryotes (Morris , 1995) and in higher plants (Einset , 1986), iP acts as may be important in conferring embryogenic a precursor of Z, probably the most active competence to tissue culture explants. For endogenous cytokinin (Kamínek et al., 1997; example, the phytohormone content of the culture Strnad, 1997). medium is a major factor regulating growth and differentiation of plant tissue in vitro only if a HORMONE CONCENTRATION VS. responsive tissue is taken as starting material (Bell SENSITIVITY et al., 1993; Somleva et al., 1995). Besides, Bögre et al. (1990) postulated that the differences Several investigations, in which a lack of between embryogenic and non-embryogenic alfalfa correlation between the endogenous hormone lines are based on their sensitivity to 2,4-D, with

R. Bras. Fisiol. Veg., 13(2):196-223, 2001 212 Víctor M. Jiménez the first line being more sensitive. It has also been barley genotypes had no effect on determining observed that 2,4-D can modulate the level of their embryogenic competence (Jiménez & auxin-binding proteins in the membranes of carrot Bangerth, 2001d). Therefore, the endogenous cell suspension cultures (Lo Schiavo et al., 1991; hormone contents seem inadequate as markers of Filippini et al., 1992; Lo Schiavo, 1995) and in embryogenic potential in those initial explants consequence may increase the sensitivity to this whose in vitro behavior is unknown. There must class of hormones. be other factors involved in determining the Guzzo et al. (1994) proposed the competence of these explants, as previously following model, which links together auxin discussed. response, asymmetric division and totipotency: In all species reported in the literature in upon environmental stimuli, cells can be made which embryogenic and non-embryogenic callus competent to respond to auxin in a morphogenetic lines could be obtained in the presence of 2,4-D, it way; if the cells have, or could be induced to was observed that the embryogenic calli contained produce the proper receptors, complete higher levels of free IAA than the non- embryogenesis would follow; if the receptors are embryogenic ones. These higher levels could be not the proper ones, only organogenesis or important in the establishment of polar auxin unorganized proliferation will occur. Even when transport, which is postulated to be determinant in the importance of cytokinin concentration in somatic embryogenesis development. In fact, as sensitivity has also been discussed, the previously mentioned, when embryogenic callus understanding of the molecular basis of cytokinin cultures lose their embryogenic competence, it is action lags behind that of other hormones (Hare & accompanied by a reduction in their endogenous van Staden, 1997). free IAA levels, down to the levels found in the non-embryogenic callus lines. In view of the CONCLUDING REMARKS results obtained, it might be postulated that these In spite of the relatively large number of high free IAA levels in the embryogenic cultures studies related to the hormonal regulation of are more likely a consequence of the embryogenic somatic embryogenesis, the results cited seem to characteristics of the tissues, than a requirement of have a low degree of concordance among them. plant tissues to be able to form embryogenic callus. As previously mentioned, there are few reports in It means that these high free IAA levels reflect the which the different hormone groups were analyzed ability of these cultures for further embryogenic systematically in the different stages of somatic development when the adequate conditionsare embryogenesis. supplied, probably by allowing the explants to With reference to induction, the initial establish an endogenous auxin gradient as soon as stage of somatic embryogenesis, it could be the adequate stimulus is given (usually a reduction concluded that, in spite of several reports that in the 2,4-D content of the culture medium). relate certain endogenous hormone levels with Although endogenous hormone levelsdo embryogenic competence of a particular genotype certainly play a role in induction and development (Rajasekaran et al., 1987a; Wenck et al., 1988; of somatic embryogenesis, the importance of Ivanova et al., 1994; Kopertekh & Butenko, 1995), sensitivity of the cells to changes in the hormone few, if any, differences were observed between concentration, both endogenous and exogenous, competent and non-competent genotypes in a may be also a key feature, and has to be further series of systematic studies, in which the same studied. There are some other factors, mentioned methodology of analysis was employed for at the beginning of this paper, which could also be determining these levels in the initial explants of determinant in induction, development and wheat and maize (Jiménez & Bangerth, 2001b, expression of somatic embryogenesis, since this 2001c). Moreover, differences found between process seems to lack a unique induction

R. Bras. Fisiol. Veg., 13(2):196-223, 2001 Regulation of in vitro somatic embryogenesis 213 mechanism. Among those factors, the role that October 1993. Legaspi City, Philippines, differences in the gene expression patterns could 1993. p. 27-36. play in conferring embryogenic capacities to a particular explant or explant section, is to be BELL, L.M.; TRIGIANO, R.N. & CONGER, B.V. considered. In this sense, certain mutant seedlings Relationship of abscisic acid to somatic of Arabidopsis can bring some light to the subject. embryogenesis in Dactylis glomerata. An example of such mutation is pickle (pkl), which Environmental and Experimental Botany, 33: 495-499, 1993. is abnormal in primary root development. When this abnormal root is excised and cultured in vitro BEN-HAYYIM, G. & NEUMANN, H. on medium lacking plant growth regulators, Stimulatory effect of glycerol on growth and somatic embryos are produced spontaneously, an somatic embryogenesis in Citrus callus effect counteracted by administration of GA (Ogas cultures. Zeitschrift für Pflanzenphysiologie , et al., 1997). Mordhorst et al. (1998) found that 110:331-337, 1983. the rate of somatic embryo formation is higher in the cultures of the primordia timing, clavata1 and BESSE, I.; VERDEIL, J.L.; DUVAL, Y.; SOTTA, clavata3 mutants when compared to the wild-type. B.; MALDINEY, R. & MIGINIAC, E. Oil These mutants are characterized by an enlarged palm (Elaeis guineensis Jacq.) clonal fidelity: shoot apical , which could be more endogenous cytokinins and indoleacetic acid in responsive to auxin signaling (Mordhorst et al., embryogenic callus cultures. Journal of 1998). Genetic approaches, which have not been Experimental Botany, 43:983-989, 1992. extensively exploited in this research field, would contribute to a better understanding of this plant BHASKARAN, S. & SMITH, R.H. Regeneration developmental course (Sugiyama, 2000). in cereal tissue culture: A review. Crop Science , 30:1328-1337, 1990. ACKNOWLEDGEMENTS BÖGRE, L.; STEFANOV, I.; ÁBRAHÁM, M.; The support of the German Academic SOMOGYI, I. & DUDITS, D. Differences in Exchange Service (DAAD) to conduct part of the response to 2,4-dichlorophenoxyacetic acid literature review is gratefully acknowledged. (2,4-D) treatment between embryogenic and non-embryogenic lines of alfalfa. In: NIJKAMP, H.J.J.; VAN DER PLAS, L.H.M. REFERENCES & VAN AARTRIJK, J. (Eds.) Progress in Plant Cellular and Molecular Biology, AMMIRATO, P.V. Organizational events during Dordrecht, Kluwer Academic Publishers, 1990, somatic embryogenesis. In: GREEN, C.E.; p. 427-436. SOMERS, D.A.; HACKETT, W.P. & BIESBOER D.D. (Eds.) Plant Tissue and Cell CARMAN, J.G. Improved somatic embryogenesis Culture. New York, Alan R. Liss, 1987, p. 57- in wheat by partial simulation of the in-ovulo 81. oxygen, growth-regulator and desiccation environments. Planta, 175:417-424, 1988. BANGERTH, F. Role of phytohormones in cell and tissue culture. In GTZ. PLANT CARMAN, J.G. Embryogenic cells in plant tissue BIOTECHNOLOGY IN TECHNICAL cultures: occurrence and behavior. In Vitro COOPERATION PROGRAMMES. Cellular & Developmental Biology, 26:746- INTERNATIONAL GTZ WORKSHOP. 6-11 753, 1990.

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