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REVIEW

REGULATION OF IN VITRO SOMATIC EMBRYOGENESIS WITHEMPHASIS 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 somaticembryogenesis , are described. Although a brief description of the effects of the addition of different plantgrowth regulators to the culture medium is given, the article is centered on the effect that endogenoushormone concentrations in the initial explants and in the tissue cultures derived from them could play inthe induction and expression of somatic embryogenesis. It is significant that few systematic studies havebeen conducted, in which different species and hormone groups were compared in cultures with andwithout embryogenic capacity. Moreover, the lack of correlation between the results presented indifferent studies indicates that the hormone content of the cultures is not the only factor involved.

ADDITIONAL INDEX TERMS: In vitro morphogenesis, embryo regeneration, embryogeniccompetence, plant growth regulators, hormones.

ABBREVIATIONS - 2,4-dichlorophenoxyacetic acid (2,4-D); abscisic acid (ABA); gibberellic acid(GA3); indole-3-acetic acid (IAA); N6-(∆2-isopentenyl) adenine (iP); N6-(∆2-isopentenyl) adenosine (iPA);proembryogenic mass (PEM); zeatin (Z); zeatin riboside (ZR).

REGULAÇÃO DA EMBRIOGÊNESE SEMÁTICA IN VITRO COMÊNFASE DO PAPEL DE HORMONIOS ENDÓGENOS

RESUMO - Neste trabalho se faz uma revisão de diversos aspectos da regulação da embriogênesessomático in vitro. Vários aspectos gerais a este fenômeno tem sido discutidos, tais como a definição determinologia, descrição de eventuais aplicações, seus estados de desenvolvimento e outros fatoresassociados com sua indução e expressão. Embora se faça uma breve descrição do efeito da adição dediferentes reguladores de crescimento ao meio de cultivo, o artigo está centrado no efeito que asconcentrações hormonais endogênas nos explantes iniciais e nos cultivos in vitro derivados deles podemter na indução e expressão da embriogênese somática. Tem de se fazer ênfase na pouca quantidade deestudos sistemáticos realizados neste tema que comparem em várias espécies e diferentes gruposhormonais em cultivos com e sem competência embriogênica. Finalmente, indica-se que a falta decorrelação entre os resultados destes poucos trabalhos parece indicar que os conteúdos hormonaisendôgenos 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: 11/06/011. CIGRAS, Univesidad de Costa Rica, 2060 San Pedro, Costa Rica. e-mail: [email protected]

Regulation of in vitro somatic embryogenesis

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INTRODUCTION

There are two alternative mechanisms bywhich an explant can regenerate an entire plant,namely organogenesis and somatic embryogenesis.Generally, in the first case, shoots and roots formsequentially and in response to appropriate cultureconditions (mainly to the type and concentration ofplant growth regulators present in the culturemedium). This type of development is alsocharacterized by the presence of vascularconnections between the mother tissue and theregenerating section (Terzi & Lo Schiavo, 1990).

On the other hand, somaticembryogenesis can be described as the process bywhich haploid or diploid somatic cells develop intostructures that resemble zygotic embryos (i.e.,bipolar structureswithout any vascular connectionwith the parental tissue) through an orderly seriesof characteristic embryological stages withoutfusion of gametes (Williams & Maheswaran, 1986;Emons, 1994; Raemakers et al., 1995). One strikingcharacteristic of the somatic embryo is itscontinuous growth resulting from the absence ofdevelopmental arrest (Faure et al., 1998). Bothprocesses, organogenesis and somaticembryogenesis, have been reported to occur in thesame explant (He et al., 1990), but originate fromparticular tissue layers or cells within explants(Osternack et al., 1999).

A number of specialized examples ofsomatic embryogenesis have been reported tooccur in vivo, from both reproductive tissues suchas the nucellus and synergid cells, and somatictissues such as somatic cells in ovules (apomixis)and leaf margins (Williams & Maheswaran, 1986;Merkle et al., 1990, and also reviewed by Sharma &Thorpe, 1995). However, somatic embryogenesis isnowadays best known as a pathway to induceregeneration from in vitro tissue cultures.

Although it is widely accepted that thefirst descriptions of in vitro somatic embryoproduction were carried out independently bySteward et al. (1958) and Reinert (1959) workingwith carrot, recently Krikorian & Simola (1999)underlined the less noticed pioneer role of HarryWaris, working with Oenanthe aquatica

(Umbelliferae). These early studies were verysignificant, because they confirmed Haberlandt’sprediction that embryos can arise from single cellsin culture (i.e., cellular totipotency) (Kiyosue et al.,1993; Höxtermann, 1997). The early history ofsomatic embryogenesis has been reviewedelsewhere (Raghavan, 1986; Halperin, 1995;Krikorian & Simola, 1999).

Since these first studies, the number ofhigher plant species from which somatic embryoscould be obtained and regenerated hascontinuously increased. This phenomenon hasbeen documented in at least 200 Gymnosperm andAngiosperm species (reviewed by Raemakers et al.,1995). Some species, however, are morerecalcitrant than others regarding both initiation ofembryogenic cultures and regeneration of plants(Rao, 1996).

Finding the right conditions to inducesomatic embryogenesis in different species andcultivars is yet, for the greater part, based on trialand error experiments (Jacobsen, 1991; Henryet al., 1994): analyzing the effect of differentculture conditions and media and modifyingespecially the type and levels of plant growthregulators. However, the role that the genotypeand its physiological condition play in this processhas seldom been studied. This is a limitation forimprovement of long-term tissue cultures, to thepoint that it restricted the development ofprotoplast fusion techniques and delayed for yearsthe development of genetic transformationtechniques beneficial for plant breeding ofmonocots (Henry et al., 1994). Only recently, byemploying modern methods of hormonalmanipulations (transgenic and habituated callus) incombination with precise and sensitive methods forhormone determination, has some progress beenachieved in this area (Bangerth, 1993). Most of thesuccess achieved so far in understanding themechanisms that govern the efficient regenerationof plants through somatic embryogenesis has beenaccomplished with model plant species and thetransfer of these new technologies to major cropspecies has been slow and difficult (Vasil, 1987).The successful induction of somatic embryos and

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subsequent recovery of viable plants is not routineor efficient for the majority of species (Merkleet al., 1995). The ability to understand themechanisms involved in the induction andexpression of somatic embryogenesis in differentspecies will increase the number of genotypescapable of regeneration by this process.

APPLICATIONS OF SOMATICEMBRYOGENESIS

Somatic embryogenesis is a veryvaluable tool for achieving a wide range ofobjectives, from basic biochemical, physiologicaland morphological studies, to the development oftechnologies with a high degree of practicalapplication.

One of the main uses of somaticembryogenesis constitutes its employment as anapproach to investigate the initial events of zygoticembryogenesis in higher plants. Perhaps theprimary reason for the limited progress inunderstanding the developmental events in plantembryos is that zygotic embryos of higher plantsconsist of several tiny cells that grow withinmaternal tissues, such as the flowers or immaturefruits, and it is quite difficult to collect sufficientembryos for biochemical, physiological andmorphological analyses of the biological eventsthat occur early in the developmental process.Somatic embryos provide a good model system bywhich such problems could be circumvented. Theknowledge of many of the events that occur duringthe early embryogenesis has resulted fromexperiments on somatic embryogenesis of a fewplant species (de Jong et al., 1993; Kiyosue et al.,1993; Zimmerman, 1993).

The mass propagation of plants throughmultiplication of embryogenic propagules is themost commercially attractive application ofsomatic embryogenesis (Merkle et al., 1990).Somatic embryogenesis has many advantages overorganogenesis in this respect: (a) it permits theculture of large numbers of ‘reproductive units’–e.g., 60,000 to 1.35 million somatic embryos perliter of medium– with the presence of both rootand shoot meristems in the same element; (b) the

modeof culture permits easy scale-up transferswith low labor inputs since embryos can be grownindividually and freely floating in liquid medium;(c) unlike shoots, somatic embryos frequentlyoriginate from single cells and the embryogeniccultures can be synchronized and purified so thatone can deal with practically pure cultures ofhomogeneous material; and, (d) plants derivedfrom somatic embryos are less variable than thosederived by way of organogenesis (Ammirato, 1987;Merkle et al., 1990; Terzi & Lo Schiavo, 1990;Osuga et al., 1999). The last point mentionedabove could be explained by an intolerance ofsomatic embryos to mutations in any of thenumerous genes that must be necessary for asuccessful completion of ontogeny (Ozias-Akins &Vasil, 1988), while vegetative meristems may bemore tolerant to mutations and epigenetic changes(Merkle et al., 1990).

Another application is in the productionof plants with different levels of ploidy; i.e.,obtaining haploid embryos by cultivating anthersand raising triploids from endosperm have beensuggested and, to a very limited extent, exploited(Terzi & Lo Schiavo, 1990). Also, success ininducing dormancy and the accomplishment oflong-term storage, together with the achievementof encapsulation of somatic embryos, has openedup the possibility for their use in the synthetic seedtechnology (Gray & Purohit , 1991; Gray et al.,1995; Litz & Gray, 1995).

The use of embryogenic callus and cellsuspension cultures, as well as somatic embryosthemselves as a source of protoplasts, has beenexploited for a range of species, taking advantageof the totipotency of these embryogenic cultures(Merkle et al., 1990). Embryogenic cultures haveproven to be especially valuable in providing asource of regenerable protoplasts in graminaceousspecies (Finch et al., 1991; Chang & Wong, 1994;Lyznik & Hodges, 1994; Funatsuki et al., 1996),Citrus species (Jiménez , 1996), and forest trees(David, 1987; McCown & Russell, 1987).

Gene transfer into embryogenic plantcells is already challenging conventional plantbreeding, and has become an indispensable tool for


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crop improvement. One of the most importantprerequisites for genetic manipulation of plantsin vitro is the ability to grow somatic cells in sterileplant growth medium and to regenerate viableplants from these cultures. Somaticembryogenesis, therefore, is a more efficientpathway for studies involving production ofgenetically transformed plants (Litz & Gray, 1995;Vicient & Martínez, 1998).

Secondary or recurrent embryogenesis,which is reported in at least 80 species (reviewedby Raemakers et al., 1995), offers a great potentialfor in vitro production of embryo metabolites, suchas lipids and seed storage proteins. However, sincethe production costs are still higher than theextraction from natural seeds, this technology isnot yet commercially viable (Merkle et al., 1990).

Finally, the embryogenic development ofsomatic cells appears to be more sensitive to theapplication of exogenous chemical compoundsthan the growth of whole plants or even calluscultures. This offers the possibility of usingin vitro screening and selection procedures toidentify plant genotypes resistant to certain factors,such as aluminum toxicity or toxins produced bypathogens (Merkle et al., 1990).

DEVELOPMENT OF SOMATICEMBRYOGENESIS

Somatic embryogenesis development hasbeen divided into two main phases, namely, theone whereby differentiated somatic cells acquireembryogenic competence and proliferate asembryogenic cells, and the phase whereby theembryogenic cells display their embryogeniccompetence and differentiate into somaticembryos. Both processes appear to be independentfrom each other and thus to be influenced bydifferent factors. The former phase, called ‘Phase0’ by Komamine et al. (1992), ‘determinationphase’ by Rao (1996) and‘induction phase’ byDodeman et al. (1997), has no direct counterpart inzygotic embryogenesis (Emons, 1994). In thiswork, the term ‘induction’ will be used todesignate the first phase and ‘expression’ for thesecond one.

The term ‘embryogenic cell’ is restrictedto those cells that have completed their transitionfrom a somatic (non-embryogenic) state to one inwhich no further exogenously applied stimuli, suchas the application of growth regulators, arenecessary to produce the somatic embryo(Komamine et al., 1992; de Jong et al., 1993). Thecells that have reached this transitional state andhave already started to become embryogenic, butthat still require exogenously applied stimuli, aredesignated as competent cells (Mordhorst et al.,1997).

Induction of somatic embryogenesis

Induction of embryogenic growth incarrot cultures, as well as in cultures of many otherspecies, appears to occurin one of two ways.Somatic embryos can form directly on the surfaceof an organized tissue such as a leaf or stemsegment, zygotic embryo, from protoplasts or frommicrospores (i.e., the cells may be considered asalready determined for embryogenic development,needing only permissive conditions to allow itsexpression). They can also form indirectly via anintermediary step of callus or suspension culture(in these cases a more complex medium should beused, including additional factors to inducededifferentiation and reinitiation of cell division ofalready differentiated cells before they can expressembryogenic competence) (Williams & Maheswaran,1986; Ammirato, 1987; Emons, 1994).

Direct and indirect somaticembryogenesis have also been considered as twoextremes of a continuum (Williams & Maheswaran,1986; Carman, 1990). Once induction ofembryogenic determined cells has been achieved,there appear to be no fundamental differencesbetween indirect and direct somatic embryogenesis(Williams & Maheswaran, 1986). Emons (1994)argued that in many systems in whichembryogenesis has been designated as indirect, theembryogenic callus is composed of young embryos(pre-embryogenic masses or (pre-)globularembryos) and that their further developmentdependson the duration of the application of theinductive stimulus. If that period is relatively

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short, the process will be direct and if it is long,then the process will be indirect. On the otherhand, in other studies, direct embryogenesis hasbeenused to describe the formation of an embryofrom a single cell without an intervening callusstage, although the embryo has arisen by means ofthe dedifferentiation of a differentiated cell withinthe explant (c.f. references in Ammirato, 1987).

In the carrot model described byKomamine et al. (1992), competent single cells(State 0) formed embryogenic cell clusters (State1) in the presence of auxin. These single cells areconsidered as predetermined for embryogenesis.During this phase, the cell clusters gained theability to develop into embryos when auxin wasremoved from the medium, leading to thedevelopment of State 1 cell clusters (Nomura &Komamine, 1985; Komamine et al., 1992).Embryogenic cells are unique: superficially theyresemble meristematic cells, though they generallyare smaller, more isodiametric in shape, havelarger, more densely staining nuclei and nucleoli,and have a denser cytoplasm (Williams &Maheswaran, 1986; Carman, 1990).

Expression of somatic embryogenesis

Once the inductionof an embryogenicstate is complete, the mechanisms of patternformation that lead to the zygotic embryo arecommon to all other forms of embryogenesis(Mordhorst et al., 1997). Thus, somatic and zygoticembryos share similar gross ontogenies, with bothtypically passing through globular, heart-shapedand torpedo-shaped stages in dicots, or globular,scutellar (transition), and coleoptilar stages inmonocots (Gray et al., 1995; Toonen & de Vries ,1996). Schiavone & Cooke (1985) described anintermediate growth stage between globular andheart-shaped embryos, that they termed the oblongembryo. Although heart- and torpedo-shapedembryos have traditionally been defined asseparate stages of the embryo development, thedistinction between them is apparently based onthe difference in size (Schiavone & Cooke, 1985).These shape-based differences have their origin inthe particular development of each of the two

major groups of flowering plants, i.e., monocotembryos initiate only a single cotyledon andconsequently do not proceed through a heart-shaped stage (Kaplan & Cooke, 1997). Yet anothertype of embryo development takes place inconifers (Tautorus et al., 1991), which includesthree stages: globular, early cotyledonary and latecotyledonary embryos (Dong & Dunstan, 2000).

In the aforementioned model ofKomamine et al. (1992), describing the earlyprocess of embryogenesis, ‘Phase 1’ is designatedas the first phase in the expression of somaticembryogenesis. This phase is induced by thetransfer of State 1 cell clusters (already induced toexpress embryogenic development) to auxin-freemedium. During this phase, cell clustersproliferate slowly and apparently withoutdifferentiation. After this phase, rapid cell divisionoccurs in certain parts of cell clusters, leading tothe formation of globular embryos (designated as‘Phase 2’). In the following phase (Phase 3),plantlets develop from globular embryos throughheart- and torpedo-shaped embryos.

Expression of somatic embryogenesismight be triggered by different factors, dependingon species, cultivar, physiological conditions of thedonor plant and so on. However, as alreadymentioned, the most common procedure is theexclusion or reduction of the auxin (mainly2,4-dichlorophenoxyacetic acid [2,4-D])concentration in the culture medium ofembryogenic cultures induced with this plantgrowth regulator.

FACTORS ASSOCIATED WITHEMBRYOGENIC COMPETENCE

To take advantage of the previouslymentioned potentialities that somaticembryogenesis offers, it is of great importance tounderstand the mechanisms underlying thetransition from a somatic or a gametophytic cell toan embryogenic cell and, in most cases, to be ableto regenerate plants from it (Mordhorst et al., 1997).At the same time, it is desirable to extend theseinduction and expression capacities to those


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species and cultivars that are recalcitrant tosomatic embryogenesis, and are also of agronomicimportance. When studying the factors involved inthe control of somatic embryogenesis, it is veryimportant to differentiate between those related tothe induction and those related to the expression ofthis event (Ermakov & Matveeva, 1994). Somaticembryogenesis induction can be spontaneous atone end of the spectrum or may demand long andcomplex treatments at the other end (Toonen & deVries , 1996). Embryogenic competence is the termused for describing the relative ease of inducingsomatic embryogenesis from tissue cultures(Carman, 1990).

Explant (non-embryogenic) cells can beinduced to an embryogenic state by a variety ofprocedures that usually include exposure to plantgrowth regulators, pH shock, heat shock ortreatment with various chemical substances.However, it is still not clear whichchanges asomatic cell must undergo in order to become anembryogenic cell capable of forming an embryo.There appears to be no single, universallyapplicable signal that renders cells embryogenic(Mordhorst et al., 1997). Moreover, in general onlya very limited number of cells in any given explantresponds by becoming embryogenic (Toonen & deVries , 1996).

Structural factors

The initiation of polarity in embryos isoften regarded as the first step in embryogenesis(Warren Wilson & Warren Wilson, 1993). In carrotand Medicago it appears that the first division hasto be asymmetric, producing two cells of differentsizes, in order to confer embryogenic competenceto the individual cells (Dudits et al., 1991). In thecase of carrot, the first division of singlesuspension cells capable of forming embryogeniccells is unequal (Komamine et al., 1992), and onlythe smaller daughter cell will ultimately developinto an embryo (de Jong et al., 1993). Komamineet al. (1992), Tsukahara & Komamine (1997) andSato-Nara & Fukuda (2000) reported a polarizedDNA synthesis in these cells. In most speciesshowing embryogenic capacity, the asymmetric

division does not form an embryo directly, butforms a proembryogenic mass (PEM), in whichonly one or a few cells can subsequently developinto an embryo (Komamine et al., 1992; Nuti Ronchi& Giorgetti, 1995). The rest of the PEM cells areprobably eliminated through a cycle ofprogrammed cell death, as observed in Norwayspruce (Filonova et al., 2000).

In a close relationship with in vitroembryogenesis, the orientation of the first celldivision has also been considered to be essential tothe establishment of the basic polarity of thezygotic embryo. However, a more extensivesurvey of the embryological literature turned up aminimum of 19 different dicotyledonous familiesin which the zygotes of at leastone species undergolongitudinal, oblique, or even variable firstdivisions instead of the expected transversedivision (Kaplan & Cooke, 1997).

In general, it can be concluded that acorrect asymmetrical first division is not anessential requirement for somatic or for zygoticembryogenesis. This does not imply that anasymmetrical distribution of intracellulardeterminants is not important in the early stages ofplant embryogenesis, but rather that such adistribution is not necessarily visibly fixed by anasymmetrical first division. There are some otherstructural factors that could influence the capacityforembryogenic induction in certain cells:microtubule organization and cell wall (cell sizeappears to play an important role in somaticembryogenesis) (Toonen & de Vries, 1996).McCabe et al. (1997) recently observed that a cell-wall antigen on cells destined to form embryossegregates asymmetrically during a formativedivision, producing one daughter cell with a cellwall antigen recognized by the antibody JIM8 andthe other without, and the epitope-free cellsultimately form somatic embryos.

Another important point is the necessityof the cells subjected to embryogenic induction, forphysical isolation from the surroundings. This isthe case with somatic embryos formed insuspension cultures. This physical isolation leadsto varying degrees of physiological isolation

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caused by loss of plasmodesmata betweensurrounding cells, interrupting symplasticcontinuity and reducing electrical coupling (WarrenWilson & Warren Wilson, 1993).

Physiological factors

Although plant growth regulators play akey role in inducing somatic embryogenesis, thereare many other factors that have been found whichcould affectthe disposition of a particular tissue toundergo somatic embryogenesis. The range ofpossible induction treatments suggests that it isunlikely that a single inducing molecule isresponsible (Toonen & de Vries, 1996).

Examples of these other factors that candirect the transition from somatic cells to cells ableto form embryo-like structures are: in Citrussuspension cultures a change in carbon sourcefrom sucrose to glycerol (Ben-Hayyim & Neumann,1983; Gavish et al., 1991; Jiménez & Guevara,1996); in carrot the NH4

+ concentration (Smith &Krikorian, 1989) and pH changes (Smith &Krikorian, 1990, 1992) in the culture medium; inAraujia sericifera the light quality (Torné et al.,2001), in Brassica microspores a temperatureshock (Pechan & Keller, 1988); also pre-treatmentof donor plants and subculture duration (Móroczet al., 1990), to name only a few factors. A moredetailed description of some of these factors wasgiven by Tisserat et al. (1979), Sharp et al. (1980),Tulecke (1987) and Harada (1999).

Culture density of cell suspensions couldbe another important factor that affects somaticembryogenesis. While a high cell density(105 cells/ml) is required for the formation ofembryogenic cell clusters from single cells(Nomura & Komamine, 1985), a lower cell density(2 x 104 cell/ml) favors the development ofembryos from embryogenic cells (Fujimura &Komamine, 1979). This may be related to thesecretion of proteins and/or other cellular factorsinto the culture medium. In fact, secreted(extracellular) and constitutive (intracellular)proteins are also considered to play a veryimportant part in induction of somaticembryogenesis (de Vries et al., 1988a,b; Gavish

et al., 1991, 1992). The addition ofarabinogalactan proteins, isolated from the culturemedium of embryogenic carrot lines and from drycarrot seeds, was capable of promoting theformation of PEMs, even in previously non-embryogenic carrot cell lines. Similar proteins, butisolated from the medium of non-embryogeniclines, acted negatively on the formation of PEMs(de Jong et al., 1993). McCabe et al. (1997) foundthat cells competent to become embryogenicrequire a soluble signal from other cells to triggersomatic embryogenesis, and postulated that signalis an arabinogalactan protein. The way in whichsecreted proteins influence somatic embryogenesisis not known, but it is reasonable to postulate thattheir function can be explained in terms of aneffect on particular cell wall polymers (van Holstet al., 1981). For a review of the role of theseproteins in this process, refer to de Jong et al.(1993), Kiyosue et al. (1993) and Schmidt et al.(1994).

Meijer et al. (1999) observed that co-culture of Arabidopsis thaliana suspension cultureaggregates interferes with the development ofcarrot somatic embryos beyond the globular stage.These authors attribute this effect to the release ofpreviously accumulated 2,4-D by the Arabidopsiscultures. Moreover, Kobayashi et al. (2000) foundthat somatic embryogenesis is strongly inhibited incultures of carrot cells when the cell density ishigh, and successfully isolated and identif ied theinhibitory factor 4-hydroxybenzyl alcohol, whichwas found to strongly inhibit the formation ofsomatic embryos when added to the culturemedium at a concentration equal to that found inhigh-cell-density cultures. Differences in thecontents of reducing sugars and starch have beenreported to be characteristic of embryogenic andnon-embryogenic calli from Medicago arborea,with higher sugar concentrations and lower starchcontent in the embryogenic cultures than in thenon-embryogenic cultures (Martin et al., 2000).

Gene expression

Plant development and differentiation areregulated directly or indirectly by changes of gene


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expression, especially during embryogenesis(Dong & Dunstan, 2000). Molecular studies onplant embryogenesis began in the 1980’s, and sincethen they have evolved in an interesting way.Initial studies of zygotic embryogenesis weregenerally designed to estimate the number ofdifferent RNAs present in developing seeds, toexamine the spatial and temporal distribution ofdistinct RNA species, to isolate and to characterizethe genes that code for abundant seed proteins,particularly the seed-storage proteins, and toidentify the cis-acting regulatory sequences andtrans-acting DNA-binding proteins that regulateexpression of seed-specific genes (Meinke, 1995).

However, the basic experimental strategyfor molecular analysis of somatic embryogenesishas mostly relied on comparing genes and proteinsbeing expressed in embryogenic and non-embryogenic cells as well as in the different stagesof embryogenesis (Rao, 1996). Gene expressionstudies during the different stages of this processhave been carried out (reviewed by Henry et al.,1994; Kawahara and Komamine, 1995; Meinke,1995; Wilde et al., 1995 and Dong & Dunstan,2000) and suggest that the number of genesspecifically expressed during these events is ratherlimited (Komamine et al., 1992; Ermakov &Matveeva, 1994; Dodeman & Ducreux, 1996;Schrader et al., 1997), and that changes in proteinpatterns are highly regulated posttranscriptionally,at the mRNA level (Komamine et al., 1992; Wildeet al., 1995). Additionally, Dodeman & Ducreux(1996) indicate that changes in hormonal levels intissue cultures may modify the synthesis of somesomatic embryogenesis-specific-proteins.Recently, Kitamiya et al. (2000) succeeded inisolating two genes that were induced afterexposure of carrot hypocotyls to highconcentrations of 2,4-D for 2 hours, a treatmentthat initiated somatic embryogenesis directly onthese explants.

Cell cultures of carrot that are growing asunorganized callus in the presence of 2,4-D havebeen used to study genetics during expression ofsomatic embryogenesis, the latter of the two phasesin which, as previously described, this process has

been divided. On the other hand, some mutationsof Arabidopsis thaliana have been used tocharacterize the induction phase, i.e., the first stageduring somatic embryogenesis.

Most genes expressed differentiallyduring somatic embryogenesis belong to the lateembryo-abundant (lea) genes. Proposed functionsfor the products of this family of genes aretheprotection of cellular structures in mature embryosduring seed desiccation and prevention ofprecocious germination of the zygotic embryosduring seed development (reviewed by Wildeet al., 1995 and Dong & Dunstan, 2000). Severalgenes expressed in carrot somatic embryos codefor secreted extracellular proteins. One geneproduct (EP1), with homology to Brassica S-locusglycoproteins, is present in nonembryogenic callusbut not in somatic embryos themselves. Anothergene that produces a lipid transfer protein (EP2)has been particularly useful as a marker forepidermal cell differentiation duringembryogenesis. The precise role of theseextracellular proteins remains to be established, butthey may be involved in the regulation of cellexpansion and the maintenance of biophysicalfeatures required for morphogenesis. Perhaps themost unexpected finding involves a secretedglycoprotein (EP3) that rescues a temperature-sensitive mutant of carrot (ts11) that fails tocomplete the transition from a globular to heartstage of somatic embryogenesis (Meinke, 1995;Sugiyama, 2000). Another group of genes that aredevelopmentally regulated in carrot suspensioncultures is constituted by the Dc3, Dc8, J4e andECP31 genes. They are expressed at differentmomentsduring embryo development and localizedin different cell groups within the PEMs andembryos (Wilde et al., 1995). Dudits et al. (1995)indicate that the gene expression is expected to bedifferent during the processes of embryogeniccommitment in primary explants or fullydifferentiated somatic cells from those that act insuspension cultures with proembryogenicstructures, such as in the case of carrot.

In recent years, there has been a shifttoward combining the tools of molecular biology

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with the power of genetics. Future research inplant molecular biology must focus not simply onisolating and characterizing large numbers of genesexpressed during plant embryo development, butalso on determining the biological significance ofthese genes by demonstrating what happens whentheir function is disrupted. This can beaccomplished either by creating transgenic plantsthat express an antisense construct or by workingwith genes that have already been disruptedthrough loss-of-function mutations (Meinke,1995).

The general strategy in genetic analysis isto use mutants either as markers of cell lineages oras vehicles for the identification of essential genes.Genetic analysis has played an increasinglyimportant role in recent studies of plantdevelopment. However, not all genes can beidentified readily by recessive loss-of-functionmutations. Genes that are duplicated in thegenome, genes that are required for early stages ofgametogenesis, and genes that perform functionsthat are redundant, nonessential, or detectable onlyin unique circumstances often escape detection inmutant screens. The power of genetics is not thatit leads to the identification of every transcriptionalunit within the genome, but rather that it allowsone to focus on genes that must be expressed inorder for growth and development to proceed in anormal manner (Meinke, 1995).

This strategy has been used more readilyto try to understand the events related to zygoticembryogenesis. Several different classes ofembryonic mutantshave been identified in higherplants. Many mutants are defective in early stagesof cell division and morphogenesis. Others fail toaccumulate pigments and storage materials duringembryonic maturation. Still others are disrupted inthe preparation for dormancy and germination.Many of these mutants are likely to be defective ingenes with housekeeping functions that firstbecome essential during embryo development.Although this information may be discouraging todevelopmental biologists interestedin finding genesthat play a direct role in the regulation of plantembryogenesis, it should be very useful for

biochemists, physiologists, and cell biologists,many of whom could use mutants defective inbasic cellular processes. Embryonic mutants alsodiffer in the initial site of gene action. The primarydefect in some embryonic mutants is limited to theendosperm tissue. Altered development of theembryo in these mutants is often an indirectconsequence of endosperm failure. The primarydefect in other mutants appears to be restricted tothe embryo proper. Although the most extensivestudies of embryonic mutants have dealt withmaize and A. thaliana, related mutants have alsobeen described in barley, carrot, rice, and peas.Most of these recessive loss-of-function mutationswere induced with chemical mutagens, X rays,transposable elements, or T-DNA fromAgrobacterium tumefaciens (Meinke, 1995).

Molecular markers

The search for markers of plantembryogenesis is an important aspect of modernplant breeding (Schel et al., 1994). Severalphysiological, biochemical and molecular markersassociated with embryogenic competence of cellshave been reported, including isozymes andmolecular markers, to be discussed in thefollowing paragraphs, and plant hormones, whichwill be discussed later.

Isozyme patterns are helpful tools for abetter understanding of the basic mechanisms ofcellular differentiation and further plantdevelopment. Apart from the classical studies inwhich isozymes were used as markers for events atthe later stages of the growing plant (e.g.,organogenesis or seed germination), during the lasttwenty years their usefulness as markers duringearly development of the plant (i.e., earlyembryogenesis) was also demonstrated.Tissueculture techniques and, more specifically, somaticembryogenesis allowed the application of isozymeanalysis to the embryogenic process, because theypermitted the availability of relatively highamounts of plant material in the desireddevelopmental stage.

Coppens & Dewitte (1990) found theesterase system to be very sensitive for detection of


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embryogenesis in barley callus before somaticembryos are formed. In carrot, two esteraseisozyme systems are differentially expressed inembryogenic and non-embryogenic cells (Chibbaret al., 1988). Tchorbadjieva & Odjakova (2001)recently found an acidic-esterase isozyme (36 kDaand an isoelectric point of 3.8) present inembryogenic suspension cultures of Dactylisglomerata but not in non-embryogenic cultures.Also, it has been observed that the pattern of acidphosphatase showed qualitative differencesbetween embryogenic and non-embryogenic coffeecallus cultures (Menéndez-Yuffá & García, 1996).An extensive review on the use of isozymes asbiochemical markers in somatic embryogenesis hasbeen already published (Schel et al., 1994).

There are several candidate genes thatcould be used as molecular markers of singlecompetent cells (Schmidt et al., 1997). One ofthese genes, the Somatic Embryogenesis Receptor-like Kinase (SERK) gene, was found to marksingle Daucus and Dactylis suspension cells thatare competent to form somatic embryos (Schmidtet al., 1997; Somleva et al., 2000).

EFFECT OF EXOGENOUSLY APPLIEDPLANT GROWTH REGULATORS ON

SOMATIC EMBRYOGENESIS

Although auxins, which are known tomediate the transition from somatic toembryogenic cells, are the agents generally used toinduce embryogenesis, the effect of other plantgrowth regulators on this phenomenon must not beoverlooked. While in Angiosperm monocots,primary embryogenesis was exclusively inducedby auxin-supplemented media, there is a largevariation of growth regulators used to inducesomatic embryogenesis in dicot species. From alist of 65 dicot species reviewed by Raemakerset al. (1995), somatic embryogenesis was inducedin 17 species on hormone-free media, in 29 specieson auxin-containing media and in 25 species oncytokinin-supplemented media. Among auxins,the most frequently used was 2,4-D (49%)followed by naphthalene acetic acid (27%), indole-3-acetic acid (IAA) (6%), indole-3-butyric acid

(6%), Picloram (5%) and Dicamba (5%). In thecase of cytokinins, N6-benzylaminopurine wasused most often (57%), followed by kinetin (37%),zeatin (Z) (3%) and thidiazuron (3%).

In those cases in which the exogenousapplication of auxins has proved to be the mostefficient treatment to induce somaticembryogenesis, further development of the existingsomatic embryos has been achieved by reducing orremoving auxin fromthe culture media. Althoughthe process of embryo induction from cells inculture is not fully understood, it is now generallybelieved that, in the continued presence of auxin, adifferential change in gene expression (probablyassociated with increased demethylation of DNA;Lo Schiavo et al., 1989) in PEMs occurs (Litz &Gray, 1995). Under these circumstances, the PEMswithin the culture synthesize all the gene productsnecessary to complete the globular stage ofembryogenesis. At that point, the PEMsalsocontain many other mRNAs and proteins whosecontinued presence generally inhibits thecontinuation of the embryogenic program. Theremoval of auxin results in the inactivation of anumber of genes so that the embryogenic programcan now proceed. The observation that somecarrot cell lines are able to develop to the globularstage, but not beyond in the continued presence ofauxin, suggests that new gene products are neededfor the transition to the heart stage and that thesenew products are synthesized only whenexogenous auxin is removed (Zimmerman, 1993).However, as stated earlier in this work, the numberof genes directly involved in development ofsomatic embryos seems to be rather limited.

The effect of the addition of other plantgrowth regulators is not so well documented. Ithas been observed that the addition of abscisic acid(ABA) inhibits the precocious germination of thesomatic embryos and allows them to mature into‘normal-shaped’ plants, as observed in grapevine(Rajasekaran et al., 1982; Goebel-Tourand et al.,1993). Bell et al. (1993) found that explants ofembryogenic genotypes of Dactylis glomerataresponded to the inclusion of ABA in the culturemedium by increasing the number of somatic

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embryos formed. Nishiwaki et al. (2000) observedthat seedlings of carrot formed somatic embryoswhen cultured on medium containing ABA, withthe number of embryos originated per explantdependent on the ABA concentration employed.Despite the wide range of physiological effects ofgibberellins, their effect when added to culturemedia, primarily as gibberellic acid (GA3), hasbeen minimal (Krikorian, 1995). Exogenousapplication of GA3 has been reported to inhibitsomatic embryogenesis and somatic embryodevelopment in several species; but it was alsoreported that this substance is required forgermination of the mature somatic embryos ifchilling is not applied (Takeno et al., 1983 andreferences therein). Even in those cases in whichthe addition of cytokinins as the sole plantregulator is effective in inducing somaticembryogenesis ,, the stimulatory effect of thesesubstances is not universal, and their additionshould often be coupled with that of auxins toobtain the desired effect (reviewed by Merkle et al.,1995).

Effect of endogenous hormones on somaticembryogenesis

It has been frequently observed thatembryogenic competent and incompetent callussections are produced in the same explant,indicating that even genetically identical cellsrespond differently to a particular stimulus, with aminority of the cells being responsive. Severalchanges should occur for reprogramming a cell toan embryogenic competent state, the first onebeing the termination of the current geneexpression patterns, permitting their replacementwith an embryogenic program, which does notoccur in all cells at the same time, and in somecells does not occur at all (Merkle et al., 1995).Anatomical and physiological differences betweenembryogenic and non-embryogenic cultures areexpressed when such changes occur. Among thesedifferences, the endogenous hormone levels shouldbe of great importance, since they regulate theprocesses of explant differentiation in culture(Grieb et al., 1997) and are postulated to be the

main difference between genotypes with variousgrades of competence (Bhaskaran & Smith, 1990).

Fot this reason the endogenous hormonelevels and their relation to the embryogeniccompetence of the explants may be the key toinduction and expression of somaticembryogenesis in recalcitrant genotypes throughamendments to the culture medium, withsubstances that may mimic the inductive condition(supplying a deficiency or counteracting anexcess), to develop or optimize in vitro protocolsfor somatic embryo production, maturation, andconversion to plantlets (Merkle et al., 1995). At thesame time, the characterization of the differencesbetween embryogenic and non-embryogenic cellsis necessary if the mechanisms involved in theinduction and maintenance of embryogeniccompetence of somatic cells are to be elucidated(Kiyosue et al., 1993).

As previously stated in this work, inmost early studies on hormonal regulation ofphysiological processes in plants, especially inin vitro cultures, more attention was paid to theeffects of the addition of plant growth regulatorsthan to the possible role of endogenoushormonal levels in the tissues. Very little isknown about the possible interactions of anendogenous phytohormone system with theexogenous growth regulators supplied to thenutrient medium. It seems probable that theobserved responses of cell culture systems, aftera growth regulator supplement, are related tosuch interactions (Neumann, 1988; Carman,1990). Evidence for this has been reported byLiu et al. (1998), who observed an accumulationof endogenous IAA in soybean hypocotylexplants after their treatment withnapththaleneacetic acid and indole-3-butyric acid(two exogenously applied auxins).

There are some reports that relate theendogenous hormone levels in the initial explants(Carnes & Wright, 1988; Kopertekh & Butenko,1995; Hess & Carman, 1998) and in the callus orcell suspension cultures derived from them (Epsteinet al., 1977; Fujimura & Komamine, 1979;Rajasekaran et al., 1982; Takeno et al., 1983; Li &


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Neumann, 1985; Rajasekaran et al., 1987b; Kiyosueet al., 1992; Michalczuk et al., 1992a; Sasaki et al.,1994; Kopertekh & Butenko, 1995; Hess & Carman,1998) to the morphogenetic competence of aparticular genotype. However, most of thesestudies were limited to only one or a fewhormones, and to one plant species. Usually,information presented in different articles can notbe put together and used to obtain a general viewof the role of endogenous hormone levels insomatic embryogenesis, because distinct genotypeswere used and also because the analytic methodswere not the same in the different studies. It isvery important to compare different plant speciessimultaneously, using the same analyticalprocedure, because from the study of a singleorganism, it is impossible to determine whether theparticular features are unique to that organism orgenerally applicable to other species (Kaplan &Cooke, 1997).

Endogenous hormone levels in the initialexplants

There are contrasting reports in relationto the effect of the endogenous hormone levels inthe initial explants on determining the ability of aparticular genotype to conduct somaticembryogenesis. Whereas differences in hormonelevels among competent and non-competent plantgenotypes have been documented, otherinvestigations reported no discrepancy, or even thatthe differences found did not account for variationsin the degree of competence.

In many cereal species, immature zygoticembryos are the primary source of explantsemployed to establish embryogenic cultures.When analyzing wheat immature embryos of twocultivars differing in their degree of competence,Kopertekh & Butenko (1995) found higher IAA andABA and lower cytokinin levels in the mostcompetent genotype. However, in a similarexperiment, but using another set of wheatgenotypes also differing in their embryogeniccompetence, Jiménez & Bangerth (2001b) foundhigher ABA levels in the competent one as theunique difference among them. The latter authors

related this finding to a reduction in the precociousgermination rate. Precocious germinationandcallus formation have been frequently reported asalternative responses of immature embryos toin vitro culture (i.e. the occurrence of onediminishes the chances of the other to occur)(Carman, 1988; Qureshi et al., 1989). Thus, areduction in precocious germination would favor,indirectly, callus formation. Working with maize,Jiménez & Bangerth (2001c) did not find anydifference in the levels of five hormones analyzedbetween two genotypes differing in their degree ofcompetence to conduct somatic embryogenesis.

In similar studiesanalyzing the wholegrains of wheat instead of isolated zygoticembryos, Hess & Carman (1998), working withwheat, found lower levels of IAA, ABA, N6-(∆2-isopentenyl) adenine (iP) and N6-(∆2-isopentenyl)adenosine (iPA) and Carnes & Wright (1988)found higher levels of total IAA and lower levelsof Z and zeatin riboside (ZR) in the mostcompetent of two maize varieties. However, it hasbeen observed that the whole kernel hormone levelpoorly reflects the levels in the immature embryossince the endosperm constitutes the majority ofkernel dry matter, and, as was demonstrated byJiménez & Bangerth (2001b, 2001c), hormonelevels in the endosperm might vary greatly inrelation to those of the immature embryos.

In three other monocot systems,Pennisetum purpureum (Rajasekaran et al., 1987a),Dactylis glomerata (Wenck et al., 1988) andMedicago falcata (Ivanova et al., 1994), in whichleaf sections were used as the initial explants,higher IAA levels were found in embryogenic thanin non-embryogenic explants. Ivanova et al. (1994)also found a positive correlation between theability to respond to a 2,4-D induction treatmentand endogenous IAA levels. The line with theshortest induction phase had the highest IAA level.The other embryogenic line needed an inductionperiod 4 to 5 times longer and had an IAA content4 times lower. In the same study, a negativerelation between the endogenous ABA level andthe embryogenic potential was observed.However, in Pennisetum purpureum leaves, higher

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levels of ABA (3- to 4-fold) were found in themore embryogenic sections than in the lessembryogenic (Rajasekaran et al., 1987b). The samegroup found that treatments which lowered ABAlevels also inhibited somatic embryogenesis, andthat this inhibition could be overcome byexogenous ABA in the medium (Rajasekaran et al.,1987a).

There are also contrasting reports inrelation to the role that endogenous gibberellinlevels in the initial explants could play indetermining the embryogenic competence. Aspreviously mentioned, Jiménez & Bangerth(2001b, 2001c) did not find any difference in theendogenous gibberellins levels (GA1,3,20) betweenembryogenic and non-embryogenic genotypes ofwheat and maize. In another study (Jiménez &Bangerth, 2001d), the same authors indicate thatdifferences in the endogenous levels of the samesubstances in two genotypes of barley did notcondition the response to the culture conditionsevaluated. Also, Rajasekaran et al. (1987a)observed that neither paclobutrazol nor the reducedlevels of gibberellins, which may have resultedfrom its application, altered the embryogeniccharacter of the explants. In contrast, Hutchinsonet al. (1997) reported that both exogenouslysupplied as well as endogenous gibberellins play anegative role in the induction of somaticembryogenesis in geranium (Pelargonium xhortorum) hypocotyl explants.

In contrast to the results obtained inalfalfa (Ivanova et al., 1994), wheat (Jiménez &Bangerth, 2001b) and maize (Jiménez & Bangerth,2001c), which indicate that the endogenouscytokinin levels in the initial explants do notapparently determine their embryogeniccompetence, Wenck et al. (1988) reported that lowendogenous levels of cytokinins werecharacteristic of leaves of Dactylis glomerata withhigh embryogenic capacity. Supporting the latterfinding, Rajasekaran et al. (1987a) reported thatboth non-embryogenic leaf regions and callus ofPennisetum purpureum contained higher levels ofcytokinins than the highly embryogenic material.Centeno et al. (1997) found similar contents of total

cytokinins in cotyledons of three Corylus avellanagenotypes with different levels of embryogeniccapacity; however, they showed a very different iP-type/Z-type cytokinin ratio. They reported higherendogenous levels of iP and lower of Z, correlatedwith higher levels of embryogenic competence,when compared to those of less competentgenotypes.

The contrasting results cited above seemto indicate that differences in the endogenoushormone levels in the initial explants of cereals arenot indicative of their embryogenic competence.Supporting this conclusion, Jiménez & Bangerth(2001d) reported marked differences in the IAAand gibberellins levels in immature zygotic barleyembryos, differences that did not affect theirdegree of competence.

Endogenous hormone levels in embryogenic andnon-embryogenic cultures

Compared to the few studies available onthe effect of the hormone status of the initialexplant on embryogenic competence, moreresearch has been carried out in relation to theendogenous hormone levels in embryogenic andnon-embryogenic callus cultures. Several reportsindicate that higher endogenous free IAA levelsare characteristic of embryogenically competentcallus lines, as observed in carrot (Li & Neumann,1985; Sasaki et al., 1994, Jiménez & Bangerth,2001a), Pennisetum purpureum (Rajasekaran et al.,1987b), sugarcane (Guiderdoni et al., 1995), wheat(Jiménez & Bangerth, 2001b) and maize (Jiménez& Bangerth, 2001c). On the other hand,Michalczuk et al. (1992a) did not find differences infree and conjugated IAA between embryogenicand non-embryogenic carrot cultures, and Besseet al. (1992) did not observe great differences inthe IAA content of callus lines of Elaeis guineensisthat differ in their embryogenic response.

Rajasekaran et al. (1987a) found that lowauxin levels coincided with high cytokinin levelsin non-embryogenic callus cultures ofP. purpureum and postulated that the lower levelsof IAA in non-embryogenic callus may be aconsequence of the uptake and accumulation of


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cytokinins, which, at optimal concentrations, havebeen shown to stimulate the formation ofisoperoxidase and IAA oxidase, enzymesresponsible for irreversible degradation of IAA(Lee, 1971). However, Jiménez & Bangerth (2001a,2001b, 2001c) could not confirm this hypothesissince they did not find any coincidence betweenlow levels of free IAA and high levels ofcytokinins in embryogenic and non-embryogeniccallus cultures of carrot, wheat and maize.

A reduction in the embryogenic capacityof the explants with time in culture underinductive conditions (specially on mediumcontaining 2,4-D) has been reported by Smith &Street (1974) and Filippini et al. (1992). Rajasekaranet al. (1987b) in P. purpureum, Kopertekh &Butenko (1995) and Jiménez & Bangerth (2001b) inwheat and Jiménez & Bangerth (2001c) in maizefound that decreased embryogenic competence wascharacterized by a reduction in the endogenousfree IAA, practically to the levels present in thenon-embryogenic lines.

Higher ABA levels in embryogeniccallus lines, when compared to non-embryogeniclines, were reported by Rajasekaran et al. (1987b) inPennisetum purpureum, Kiyosue et al. (1992) andJiménez & Bangerth (2001a) in carrot, Guiderdoniet al. (1995) in sugarcane and Jiménez & Bangerth(2000) in grapevine. Kiyosue et al. (1992)postulated that high endogenous ABA levels couldbe necessary to induce or maintain somaticembryogenesis in carrot. Contrary to these results,Etienne et al. (1993) found that high levels of ABAaccumulated in non-embryogenic calli of Heveabrasiliensis, but remained at a low level in theembryogenic callus. These latter authorspostulated that high ABA levels are incompatiblewith the initiation and then the development ofsomatic embryos.

No defined pattern was observed in theendogenous levels of gibberellins when comparingembryogenic with non-embryogenic calluscultures. While Jiménez & Bangerth (2001a) incarrot, Jiménez & Bangerth (2001b) in wheat andJiménez & Bangerth (2000) in grapevine did notfind any difference, Jiménez & Bangerth (2001c)

found higher gibberellin levels in embryogenicmaize lines and, in contrast, Noma et al. (1982)related high levels of polar gibberellins (probablyGA1) to an absence of embryogenic competence incarrot.

Concerning the endogenous levels ofcytokinins, Ernst et al. (1984) and Ernst &Oesterhelt (1984, 1985), working with anise,concluded that the cytokinin levels in the calluscultures seemed to be more related to the growthrate of the calli than to their embryogeniccompetence. Similar conclusions were postulatedby Jiménez & Bangerth (2000) in grapevine.However, Rajasekaran et al. (1987a) found levels ofcytokinins in non-embryogenic callus at least twotimes higher than in embryogenic callus, after 10days of culture of Pennisetum purpureum leafexplants. Guiderdoni et al. (1995) reported higherlevels of iP and iPA in embryogenic calli than inthe non-embryogenic calli of sugarcane, theopposite for Z, andno differences in the ZR levels.

Habituated cultures and endogenous hormones

Habituated cultures are those that canproliferate in a culture medium without thesupplement of exogenous plant growth regulators(Meins, 1989). There is some controversy inrelation to the hormone physiology of habituatedand non-habituated cells. While similarconcentrations of hormones in both habituated andnon-habituated cell types have been reported(Kevers et al., 1981), higher hormoneconcentrations were also found in habituated thanin non-habituated cells (du Plessis et al., 1996 andreferences therein). It has been argued thathabituation may be due to the elevated levels ofcytokinins in the habituated cells (du Plessis et al.,1996). However, Das and Saha (1995), analyzingthe endogenous cytokinin levels in habituated andnon-habituated nucellar callus of sweet orange,found higher levels of Z, ZR and Z glucoside in thesecond callus type.

It was previously mentioned thatembryogenic callus cultures developed on culturemedium containing 2,4-D are characterized byhigher endogenous IAA contents than those of the

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non-embryogenic cultures. Since Michalczuk et al.(1992b) postulated that the 2,4-D in the culturemedium is the cause of this increase in theendogenous IAA levels, it is conceivable to thinkthat in the habituated cultures, grown on ahormone free medium, this increase does notoccur. In grapevine, Jiménez & Bangerth (2000)reported that competent callus cultures did notshow this characteristic pattern of higher IAAlevels, instead the levels were similar inembryogenic and non-embryogenic cultures.Working with habituated Citrus callus and cellsuspension cultures, Jiménez et al. (2001) foundthat the treatment that stimulated the furtherdevelopment of the somatic embryos alsostimulated auxin and cytokinin accumulation.However, these authors did not make comparisonsbetween embryogenic and non-embryogeniccultures.

Limanton-Grevet et al. (2000) analyzedthe endogenous levels of IAA, ABA, Z, ZR, iP andiPA in eight embryogenic habituated callus linesderived from six different Asparagus officinalisgenotypes, maintained on hormone-free mediumfor more than one year. Even when a greatvariaton in the intensity of the secondaryembryogenesis was observed between lines, thiscould not be related to the hormone metabolism,since no significant differences were foundbetween distinct embryogenic lines.

PROPOSED ROLE FOR EACH HORMONEGROUP DURING SOMATIC

EMBRYOGENESIS

Although the endogenous hormone levelsby themselves do not account for all thephenomena observed during induction anddevelopment of somatic embryogenesis, a generalview of the importance of each hormone group canbe postulated.

Auxin is considered to be the mostimportant hormone in regulating somaticembryogenesis (Cooke et al., 1993). Both theendogenous contents and the application ofexogenous auxins are determin ing factors duringthe induction and expression phases of somatic

embryogenesis. It has been reported that theculture of explants in 2,4-D-containing medium,the classic induction treatment for many species,increases the endogenous auxin levels in theexplants (Michalczuk et al., 1992b).

It has also been observed that polartransport of auxin is essential for the establishmentof bilateral symmetry during embryogenesis indicotyledonous somatic (Schiavone & Cooke, 1987)and zygotic (Liu et al., 1993) embryos, and morerecently it was also demonstrated formonocotyledonous zygotic embryos (Fischer &Neuhaus, 1996). For this gradient to be established,relatively high levels of free IAA may be necessaryin the competent tissues. However, once thestimulus for the further development of the somaticembryos is given (i.e., through reduction orremoval of 2,4-D from the culture medium), thoselevels must be reduced, to allow the establishmentof the polar auxin gradient. If the levels areextremely low or high, or if they do not diminishafter the induction treatment, the gradient cannotbe formed, and thus somatic embryogenesis cannotbe expressed. Indoleacetylaspartate as been foundin high levels in embryogenic callus of ‘Shamouti’orange (Epstein et al., 1977), and it was suggestedthat through this mode of IAA conjugation, auxinlevels in embryogenic callus are reduced to a levelinducive to active embryogenesis.

Endogenous levels of ABA also appearto be significant in some monocots for initiation ofembryogenic cultures (Bhaskaran & Smith, 1990).Its role as an inhibitor of precocious germinationwhen immature zygotic embryos are used as initialexplants, thus favoring indirectly callus formation,was discussed by Jiménez & Bangerth (2001b).The role of ABA in somatic embryogenesis may beexerted through regulation of certain genes (e.g.,DC8) that are thought to be involved in desiccationand maturation phases of embryogenesis(Hatzopoulos et al., 1990). Rajasekaran et al.(1987a,b) proposed that ABA could exert its roleon somatic embryogenesis by regulatingcarbohydrate metabolism, via inhibition of α-amylase activity. There are several instanceswhere applied ABA has been shown to stimulate


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cell division and DNA synthesis, callus productionand shoot morphogenesis, and to inhibit peroxidaseactivity, an enzyme that, as previously mentioned,causes an irreversible degradation of IAA(Rajasekaran et al., 1987a and references citedtherein). Senger et al. (2001), using a homozygoustransgenic line of Nicotianaplumbaginifoliaconstitutively expresing an anti-ABA single chain fragment variable antibody inthe endoplasmic reticulum, clearly demonstratedthat ABA is important for the formation of pre-globular structures.

The role of exogenous and endogenousgibberellins in somatic embryogenesis has not beenstudied in detail. The results of different reportsseem to indicate a minor role for endogenousgibberellin levels, both in the initial explants andthe callus cultures (Jiménez & Bangerth,2001a,b,c,d).

There is support for the concept thatcytokinins , in general, are important during theinitial cell division phase of somaticembryogenesis, but not for the later stages ofembryo development and maturation in carrot(Fujimura & Komamine, 1980), anise (Ernst et al.,1984; Ernst &Oesterhelt, 1985) and orchardgrass(Wenck et al., 1988). This suggests that cytokininsmay have a role in cell division, but not in embryodifferentiation (Danin et al., 1993). Whenanalyzing the individual role of the iP- and Z-typecytokinins, Centeno et al. (1997) postulated that therole the first group in the embryogenic responsemight be as biosynthetic precursors to supply thelarge amount of active cytokinins required for thestimulation of cell division prior to somaticembryogenesis. In the general proposed pathwayof cytokinin biosynthesis in prokaryotes (Morris ,1995) and in higher plants (Einset , 1986), iP acts asa precursor of Z, probably the most activeendogenous cytokinin (Kamínek et al., 1997;Strnad, 1997).

HORMONE CONCENTRATION VS.SENSITIVITY

Several investigations, in which a lack ofcorrelation between the endogenous hormone

concentration and the response of the explants wasobserved, led Trewavas (1981) to postulate that thesensitivity of the tissues to a change in thehormone concentration is more important than thechange in the concentration itself. This proposalgenerated a lot of controversy at that time, but itwas supported by several observations, such as thefact that immature flowers and fruits did notrespond to ethylene application, but the matureones did (Davies, 1995).

Several reports, such as those in whichno differences between the endogenous levels ofmost hormones evaluated in the initial explants ofcompetent and non-competent genotypes werefound (Jiménez &Bangerth, 2001b, 2001c) or thatin which differences in the hormone levels did notseem to play any role in determining themorphogenetic capacity of the explants (Jiménez &Bangerth, 2001d), demonstrate that there are,certainly, other factors, besides the endogenouslevels of plant hormones, that determine thecompetence of the tissues to develop embryogeniccells. This kind of result seems to support thepostulate of Trewavas; however, now it is widelyaccepted that both the sensitivity of the tissues tothe plant hormones and the concentration of thehormones, are important and modulate theresponses observed in most cases. Sensitivity hasbeen shown to contribute in a higher degree to, atleast, gravitropic responses, vascular tissueregeneration, cambial growth, seed dormancy,germination, cell division, cell extension, ripening,senescence, abscission, stomatal aperture,flowering and amylase formation (reviewed byTrewavas , 1991).

There is some evidence that sensitivitymay be important in conferring embryogeniccompetence to tissue culture explants. Forexample, the phytohormone content of the culturemedium is a major factor regulating growth anddifferentiation of plant tissue in vitro only if aresponsive tissue is taken as starting material (Bellet al., 1993; Somleva et al., 1995). Besides, Bögreet al. (1990) postulated that the differencesbetween embryogenic and non-embryogenic alfalfalines are based on their sensitivity to 2,4-D, with

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the first line being more sensitive. It has also beenobserved that 2,4-D can modulate the level ofauxin-binding proteins in the membranes of carrotcell suspension cultures (Lo Schiavo et al., 1991;Filippini et al., 1992; Lo Schiavo, 1995) and inconsequence may increase the sensitivity to thisclass of hormones.

Guzzo et al. (1994) proposed thefollowing model, which links together auxinresponse, asymmetric division and totipotency:upon environmental stimuli, cells can be madecompetent to respond to auxin in a morphogeneticway; if the cells have, or could be induced toproduce the proper receptors, completeembryogenesis would follow; if the receptors arenot the proper ones, only organogenesis orunorganized proliferation will occur. Even whenthe importance of cytokinin concentration insensitivity has also been discussed, theunderstanding of the molecular basis of cytokininaction lags behind that of other hormones (Hare &van Staden, 1997).

CONCLUDING REMARKS

In spite of the relatively large number ofstudies related to the hormonal regulation ofsomatic embryogenesis, the results cited seem tohave a low degree of concordance among them.As previously mentioned, there are few reports inwhich the different hormone groups were analyzedsystematically in the different stages of somaticembryogenesis.

With reference to induction, the initialstage of somatic embryogenesis, it could beconcluded that, in spite of several reports thatrelate certain endogenous hormone levels withembryogenic competence of a particular genotype(Rajasekaran et al., 1987a; Wenck et al., 1988;Ivanova et al., 1994; Kopertekh & Butenko, 1995),few, if any, differences were observed betweencompetent and non-competent genotypes in aseries of systematic studies, in which the samemethodology of analysis was employed fordetermining these levels in the initial explants ofwheat and maize (Jiménez & Bangerth, 2001b,2001c). Moreover, differences found between

barley genotypes had no effect on determiningtheir embryogenic competence (Jiménez &Bangerth, 2001d). Therefore, the endogenoushormone contents seem inadequate as markers ofembryogenic potential in those initial explantswhose in vitro behavior is unknown. There mustbe other factors involved in determining thecompetence of these explants, as previouslydiscussed.

In all species reported in the literature inwhich embryogenic and non-embryogenic calluslines could be obtained in the presence of 2,4-D, itwas observed that the embryogenic calli containedhigher levels of free IAA than the non-embryogenic ones. These higher levels could beimportant in the establishment of polar auxintransport, which is postulated to be determinant insomatic embryogenesis development. In fact, aspreviously mentioned, when embryogenic calluscultures lose their embryogenic competence, it isaccompanied by a reduction in their endogenousfree IAA levels, down to the levels found in thenon-embryogenic callus lines. In view of theresults obtained, it might be postulated that thesehigh free IAA levels in the embryogenic culturesare more likely a consequence of the embryogeniccharacteristics of the tissues, than a requirement ofplant tissues to be able to form embryogenic callus.It means that these high free IAA levels reflect theability of these cultures for further embryogenicdevelopment when the adequate conditionsaresupplied, probably by allowing the explants toestablish an endogenous auxin gradient as soon asthe adequate stimulus is given (usually a reductionin the 2,4-D content of the culture medium).

Although endogenous hormone levelsdocertainly play a role in induction and developmentof somatic embryogenesis, the importance ofsensitivity of the cells to changes in the hormoneconcentration, both endogenous and exogenous,may be also a key feature, and has to be furtherstudied. There are some other factors, mentionedat the beginning of this paper, which could also bedeterminant in induction, development andexpression of somatic embryogenesis, since thisprocess seems to lack a unique induction


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mechanism. Among those factors, the role thatdifferences in the gene expression patterns couldplay in conferring embryogenic capacities to aparticular explant or explant section, is to beconsidered. In this sense, certain mutant seedlingsof Arabidopsis can bring some light to the subject.An example of such mutation is pickle (pkl), whichis abnormal in primary root development. Whenthis abnormal root is excised and cultured in vitroon medium lacking plant growth regulators,somatic embryos are produced spontaneously, aneffect counteracted by administration of GA (Ogaset al., 1997). Mordhorst et al. (1998) found thatthe rate of somatic embryo formation is higher inthe cultures of the primordia timing, clavata1 andclavata3 mutants when compared to the wild-type.These mutants are characterized by an enlargedshoot apical meristem, which could be moreresponsive to auxin signaling (Mordhorst et al.,1998). Genetic approaches, which have not beenextensively exploited in this research field, wouldcontribute to a better understanding of this plantdevelopmental course (Sugiyama, 2000).

ACKNOWLEDGEMENTS

The support of the German AcademicExchange Service (DAAD) to conduct part of theliterature review is gratefully acknowledged.

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