REVIEW

Molecular and cellular approaches to the analysis of plant embryo

development from somatic cells in vitro

DfiNES DUDITS, LASZL6 BOGRE and JANOS GYORGYEY

Institute of Plant Physiology, Biological Research Center, Hungarian Academy of Sciences, 6701 Szeged, P.O. Box 521, Hungary

Introduction

During the normal life cycle of higher organisms,including plants, embryogenesis is initiated by sperm-eggcontact at fertilization. The start of the developmentalprogram is a consequence of changes due to fertilizationevents as well as activation of embryogenic events withwell-defined timing (see review by Epel, 1990). Differen-tiation of totipotent embryogenic cells proceeds accordingto a pre-set program and terminally differentiated cellsare formed. In contrast to other eukaryotes, the differen-tiation program in plants is flexible and strikingly so:almost any fully differentiated plant cell can becomeembryogenic under denned conditions. Generation of atotipotent state in somatic cells with competence forreinitiation of the whole developmental cycle throughembryogenesis is a remarkable biological phenomenon. Itrelies on the reprogramming of the gene expressionpattern in the cells and on triggering the cascade ofstructural embryogenic changes as in zygotic embryodevelopment. Since the first observation of somaticembryo formation in carrot cell suspension cultures byStewards et al. (1958) and Reinert (1959), the potential forasexual embryogenesis has been shown to be character-istic of a wide range of tissue culture systems from bothdicot and monocot plants (Ammirato, 1983). Embryosdeveloped from single somatic cells in vitro can beregenerated into whole plants. Therefore, somatic embryo-genesis provides a bridge between the cellular and plantlevels and opens the use of cultured cells in geneticmanipulation experiments such as somatic hybridizationor DNA transformation (reviewed by Dudits, 1987).Despite the large number of empirical observations aboutembryogenesis in various plant tissues and cells themolecular and cellular basis of this unique, unusualdevelopmental pathway is poorly understood. The presentreview aims to outline a general concept of the central roleof hormone-induced cell divisions in resetting the develop-mental program and in induction of the embryogenic statein somatic plant cells.

Synthetic auxins as key factors In establishmentof embryogenic tissue culture systems

As a routine procedure, tissues or single cells from variousplant organs are placed onto synthetic culture mediumJournal of Cell Science 99, 475-484 (1991)Printed in Great Britain © The Company of Biologists Limited 1991

supplemented with plant hormones such as auxins,typically 2,4-dichlorophenoxyacetic acid (2,4-D). Cytoki-nin can also be an optional component of the culturemedium. Sensitive cells of primary explants, frequentlyaround the wounded area, respond to the in vitroconditions by proliferation, and actively dividing tissuesare formed. These so-called dedifferentiated callus tissuescan be propagated both in liquid culture and on the surfaceof solid agar culture medium. In this heterogeneous cellpopulation only a limited number of cells exhibitsembryogenic potential and this fraction is highly variableamong plant species. It depends on the genotype and tissueorigin of the primary explants (Ammirato, 1983; Brownand Atanassov, 1985). Carrot (Daucus carota L.) cellsuspension cultures are the most extensively studiedembryogenic systems and they are also considered to bethe ideal model for studies of the molecular basis ofsomatic embryogenesis (Borkird et al. 1988; Komamine etal. 1990; Racusen and Schiavone, 1990). It is generallyaccepted that in carrot cultures the embryogenic state isalready induced when the cell line is established and theproembryogenic mass (PEM) is propagated in the presenceof 2,4-D (Fig. 1). In addition to the crucial role of theexogenous auxins in generating embryogenic potential,2,4-D itself inhibits the progression of embryo develop-ment in carrot multicellular colonies. Therefore thecompletion of embryogenesis requires hormone-free cul-ture conditions or low cell density (Sung and Okimoto,1981). In carrot cultures small, round single cells withdense cytoplasm can be identified and separated byphysical means (Nomura and Komamine, 1985). Thisfraction of so called State 'O' cells requires 2,4-D treatmentto divide, to become embryogenic and to form embryosfrom this defined cell type in carrot suspension culturewith high frequency (Komamine et al. 1990).

Owing to the proembryogenic nature of carrot suspen-sion cultures, a comprehensive study of the activationevents at initiation of the embryogenic development insomatic cells requires additional experimental systems.For example, selected genotypes of various Medicagospecies can be used to produce in vitro cultures in that thetime of embryogenic induction can be controlled (Fig. 1).Fig. 2A shows dedifferentiated alfalfa microcallus suspen-sion (MCS) grown in the presence of a weak auxin such as

Key words: embryo development, somatic plant cells, fertiliz-ation, signal transduction, cell cycle control.

473

CARROT CULTURE ALFALFA CULTURE

Initiation of callus tissues

2,4-D NAA + KIN

Long-term propagationin liquid culture

^

Proembryogenic mass (PEM) Microcallus suspension (MCS)

2,4-D

removal of 2,4-D

NAA + KIN

2,4-D shock(treatment with

1 00/AM for 1 hour)

EMBRYO DEVELOPMENTunder hormone-free conditions

GLOBULAREMBRYO

HEART-SHAPEDEMBRYO

TORPEDO-SHAPEDEMBRYO

Fig. 1. Key steps in initiation of embryogenic potential and embryo development in carrot and alfalfa cultures. 2,4-D,2,4-dichlorophenoxyacetic acid; NAA, naphthalene acetic acid; KIN, kinetin.

474 D. Dudits et al.

Fig. 2. Trigger of somatic embryogenesis in alfalfa microcallus suspension (MCS) by a short pulse of auxin shock.(A) Undifferentiated multicellular colonies in MCS in the presence of 15 IOA naphthalene acetic acid and 10 /ai kinetin. (B) Mixtureof somatic embryos with different developmental state in MCS treated with 100/JM 2,4-dichlorophenoxyacetic acid for l h andgrown in hormone-free medium for 28 days'. (C) Separated fraction of early globular-stage somatic embryos. (D) Late torpedo-stageembryos.

naphthalene acetic acid (NAA). Only a very short pulse oftreatment (a few minutes up to few hours) with a strongauxin (2,4-D) is sufficient to initiate organized growth inmulticellular structures of MSC and to induce subsequentformation of embryos in hormone-free culture medium(Fig. 1 and Fig. 2 B,C,D).

Alfalfa also offers a uniform single cell system foranalyzing the dramatic developmental switch under invitro conditions. If leaf tissues are treated with cell wall-degrading enzymes, protoplasts can be released from fullydifferentiated mesophyll cells. In protoplast culture me-dium in the presence of auxin (2,4-D) and cytokinin (zeatinriboside) resynthesis of the cell wall is followed byinitiating cell division and embryos can be formed fromsingle cells without the intervening stage of callusformation (Kao and Michayluk, 1980; Song et al. 1990).The frequency of this direct embryo formation can beincreased by exposing the protoplast-derived cells to low-voltage electrical fields (Dijak et al. 1986).

Synthetic auxins such as 2,4-D are essential constitu-ents of culture media used for production of embryogeniccultures of monocotyledonous plants including rice(Abdullah et al. 1986), wheat (Vasil et al. 1990) and maize(M6rocz et al. 1990).

Components of signal transduction during auxin-mediated cellular changes

In view of the results from extensive experimentation witha variety of embryogenic plant tissue cultures exogenouslyapplied auxins can be identified as one of the key inducersof embryogenic development in somatic plant cellscultured in vitro. Only a fraction of the cells appears to becapable of an embryogenic response. Differences in auxinsensitivity of the cells can be suggested as a limiting factorin the complex interaction between cells and synthetichormones. Indeed, the detailed comparison betweenembryogenic (A2) and non-embryogenic (R15) clones fromthe same genotype of alfalfa (Medicago varia cv. Rambler)has revealed considerably increased sensitivity to 2,4-D inprotoplast-derived cells or root explants of the embryo-genic genotype (Bogre et al. 1990). Embryogenesis occursin tissues or colonies grown in the presence of 2,4-D atconcentrations that already inhibit the growth of callustissues. The minimum concentration or the duration of2,4-D treatment required for inductive effect is different invarious genotypes and species. Induction of cell division asa 2,4-D response can result in unorganized callus growthor well-coordinated pattern-forming polarized growth ofembryo development. These two pathways should beexperimentally separated in order to reveal their basicmolecular and cellular differences (see Fig. 3.).

Considering auxin treatment as an external stimulus,one should evaluate the possible analogy between signaltransduction systems in fertilized eggs and in 2,4-D-treated somatic cells. As listed by Epel (1990), membranedepolarization, polyphosphoinositide hydrolysis, Ca2+ re-lease, Na+, H+ exchange, pH increase, elevated oxygenconsumption and H2O2 production are the major, charac-teristic changes in sea-urchin embryos after sperm-eggbinding. Considerable similarity becomes apparent whenone compares these early events in fertilization with therapid cellular responses to auxins (reviewed by Brummeland Hall, 1987) or to wounding (reviewed by Davies, 1987).Some of these similarities are discussed as follows.

The primary consequence of auxin binding to itsreceptor is hyperpolarization of the membrane, which ismediated by a plasmamembrane H+-ATPase (Barbier-Brygoo et al. 1989). Several lines of evidence have clearlyproved the presence of auxin-binding proteins or theexistence of auxin receptors in the plasmalemma and inendoplasmic reticulum (Shimomura et al. 1988; Bariber-Brygoo et al. 1989; Inohara et al. 1989). Auxin-bindingproteins versus auxin receptors are discussed by Klambt(1990). The primary structure of these proteins wasdetermined by sequence analysis of the correspondingcDNAs (Hesse et al. 1989; Inohara et al. 1989; Tillmann etal. 1989). Auxin sensitivity could also be related to theapparent abundance of auxin-binding protein on proto-plast plasmalemma (Barbier-Brygoo et al. 1990). Atpresent we are not aware of any comparative study onauxin receptors in embryogenic and non-embryogeniccells. However, embryo formation could be achieved in thenon-responsive R15 alfalfa plants after introduction andexpression of rolB and rolC genes of Agrobacteriumrhizogenes (Bogre et al. unpublished). These bacterialgenes can cause elevation of auxin sensitivity in trans-genic plants (Shen et al. 1988).

In addition to the detection of auxin-induced alterationsin both cytoplasmic and cell wall pH (see Brummel andHall, 1987), experiments have been done with woundedcarrot explants that show that by using culture mediumcontaining 1-5 DIM NH4"1" at pH4 one can initiate andmaintain the preglobular stage in proembryogenic cul-tures without the application of any exogenous hormone;in these cultures increasing the pH will start thesubsequent phase of embryo development (Smith andKrikorian, 1990).

Involvement of phosphatidylinositol (Ptd Ins) cycle intransmembrane signalling in higher plants has becomemore evident from an increasing number of studies(reviewed by Morse et al. 1989; Guern et al. 1990). Thesestudies have demonstrated that auxins such as indole-acetic acid (IAA) or 2,4-D stimulate the hydrolysis ofPtdIns(4,5)P2 (phosphatidylinositol 4,5-bisphosphate) andthe release of InsP2 and InsP3 (Morr6 et al. 1984; Ettlingerand Lehle, 1988; Zbell and Walter-Back, 1988). The role ofInsP3, which increases the concentration of cytosolic freeCa2+, has been shown by detection of Ca2+ release fromvacuolar membrane vesicles (Ranjeva et al. 1988; Schu-maker and Sze, 1987). The other components of signallingthat are independent of or linked to the Ptdlns cycle arethe GTP-binding proteins (reviewed by Guern et al. 1990).Evidence for the existence of G-proteins in higher plantswas provided by the use of antisera raised against asynthetic oligopeptide representing a highly conservedregion of mammalian a--subunits of G-proteins (Blum et al.1988). On the basis of reports of the reciprocal effect ofauxin and GTP on their membrane binding or onsynergistic effect of auxin and GTP on phospholipase C(reviewed by Guern et al. 1990), GTP-binding proteins canbe considered as functional components in the realizationof auxin effects.

The central role of alterations in free Ca2+ levels duringfertilization and embryogenic development has beendemonstrated in animals (Speksnijder et al. 1989) andhigher plants (Timmers et al. 1989). The effect of calciumions as a second messenger is frequently mediated bycalcium-binding protein such as calmodulin (Kelly, 1984).The Ca2+-calmodulin complex can regulate enzymeactivities either directly, or indirectly through proteinphosphorylation (Poovaiah et al. 1987).

Embryogenesis from somatic plant cells 475

Callus

t 2,4-D Low

k

I 9 4-n Hinh

Fig. 3. Auxin concentration-dependent equal and unequal cell division inprotoplast-derived alfalfa leaf mesophyll cell as initiation of non-embryogenicand embryogenic developmental pathways.

Somatic embryos

476 D. Dudits et al.

Reactivation of the cell cycle as a prerequisitefor embryogenic induction

The similarities in the components of the signal transduc-tion pathway between fertilized eggs and auxin-treatedsomatic plant cells extends also to the next phase ofdevelopment. The initiation of DNA synthesis and the celldivision are key events in both systems. They areaccompanied by essential alterations in the expression of aset of genes and subsequently by coordinated changes incellular functions. As far as the cell cycle parameters areconcerned, only limited information is available fromstudies on embryogenic plant cells. When cultured carrotcells are induced to undergo embryogenesis, the celldoubling time decreases (Warren and Fowler, 1978).Fujimura and Komamine (1980) have reported rapidcycling prior to the formation of carrot globular stageembryos with a doubling time of 6.3 h. On the basis of thespatial distribution of cells undergoing DNA synthesis, apolarity was detected in developing early carrot embryos(Komamine et al. 1990). In studies on the significance ofreactivation of cell division and cell cycle regulation insomatic embryogenic plant cells, leaf mesophyll protoplastcultures can offer a relatively uniform, synchronousexperimental system. It is generally observed that freshlyisolated protoplasts are in Gi phase (Wernicke et al. 1990).Depending on the position of cells in wheat leaves,different concentrations of 2,4-D are required for stimu-lation of the Gi-S transition (Wernicke and Milkovits,1987). After resynthesis of the cell wall the protoplast-derived cells start to divide under the influence of auxinssuch as 2,4-D. Comparison of the time course of [3H]thymi-dine incorporation and the frequency of dividing cells hasclearly indicated an earlier reactivation in 2,4-D-treatedembryogenic (A2) cells than in cells derived from a non-embryogenic genotype (R15) (see Bogre et al. 1990). Thesepreliminary observations also emphasize the need fordetermination of basic elements in the regulation of thecell cycle in order to understand hormone-dependent celldivision, differentiation and somatic embryo developmentin higher plants.

The present models for the molecular mechanism of cellcycle control in eukaryotes (reviewed by Hunt, 1989; Blow,1989; Draetta, 1990; Solomon et al. 1990) describe p34cdci

protein kinase as a key regulatory element with acomplex, phosphorylation-dependent interaction with cyc-lins. The p34cdc2 kinase phosphorylates many differentsubstrates including lamins, histone HI, cyclins, RNApolymerase II (for review, see Moreno and Nurse, 1990).Murray and Kirschner (1989) suggested different regulat-ory pathways based on alteration of the active and inactiveforms of the maturation promoting factor (MPF) inembryogenic and somatic cells. Recently, various com-ponents of this control system have been proved to bepresent in plant cells. Antisera specific for pM0402, such asanti-PSTAIR peptide or anti-cdc2 Mab-J4 antibodies,recognize a 34xlO3Mr protein in plant cell extracts (Johnet al. 1989; Feiler and Jacobs, 1990; Hirt et al. 1991). TheHI kinase activity of the p34cdc2 protein was detected inpea and alfalfa (Feiler and Jacobs, 1990; Bak6 et al. 1991).Further evidence for the existence of a plant cdc2 proteinkinase has been obtained by isolation of a cDNA clone thatappears to code the alfalfa homolog of the cdc2 gene. ThiscDNA (cdc2Ms) encodes a protein with the characteristicamino acid sequence elements required for cdc2 kinasefunction. This function was proved by complementation ofa temperature-sensitive cdc2 mutant in fission yeast by

the plant cDNA (cdc2Ms) (Hirt et al. 1991). Northernanalysis revealed that the level of expression of the alfalfacdc2 gene was significantly increased by 2,4-D treatmentof a microcallus suspension (MCS) culture. The amount ofcdc2Ms transcripts was higher at early stages of somaticembryo development (Hirt et al. 1991). Amounts of thep34cdc2 protein, measured by PSTAIR peptide antibodies,were also observed at higher levels in wheat leaf segmentswith dividing cells than in regions with non-dividing,differentiated cells (John et al. 1990).

In searching for other components of the maturationpromoter factor it was found that anti-cyclin B (cdcl3)antibodies cross-react with a 62 x 103 MT protein in alfalfaprotein extract from cultured cells (Bak6 et al. 1991). Thisanalysis has also demonstrated the formation of a complexbetween the 62xlO3Mr protein and the plant p34cdc2

kinase. The cell cycle-dependent changes in p34cdc2 HIkinase activity and the presence of the phosphorylatedform of 62 x 10 Mr protein detected by in vitro phosphoryl-ation of protein extracts from protoplast-derived alfalfacells is shown in Fig. 4. For this analysis pl3BUCl-Sepharose was used, which can bind different forms of thep34cdc2_Cyclin complex (Brizuela et al. 1987). As shown bythe autoradiogram in Fig. 4, phosphorylation activity wasfound in protein extract from 3-day-old protoplast cultureafter pl38ucl-Sepharose chromatography. This sharpincrease in p34cdc2 HI kinase activity coincides with thepeak of [3H]thymidine incorporation. Detection of a Gi-Sphase-related complex that includes p34cdc2 kinase duringculture of alfalfa protoplast-derived cells is in agreementwith other evidence for the existence of a p34 kinaseactive at the Gi-S phase boundary in other systems(McVey et al. 1989; Giordano et al. 1989; Furukawa et al.1990).

In the complex network consisting of protein kinasesand multiple phosphoproteins, signals can be transmittedor amplified from receptor molecules to structural orregulatory proteins in the nucleus. The various Ca2+- orcalmodulin-dependent protein kinases detected in differ-ent plants (Harmon et al. 1987; Bogre et al. 1988; Klucisand Polya, 1988; Olah et al. 1989) are possible constituentsof this signal transduction system.

It is expected that the initiation and maintenance of thecell cycle under the influence of plant hormones isreflected by changes in transcription of cell cycle-dependent genes. In the case of the replication-dependenthistone genes a multicomponent regulatory mechanismwas described, from studies on various eukaryotic cells(reviewed by Schumperli, 1988). However, histone genescan be expressed replication-independently or in a tissue-specific manner (Old and Woodland, 1984). The recentprogress in identification of histone H3 variants on thebasis of electrophoretic characteristics of histone H3proteins (Waterborg et al. 1987) and nucleotide sequence ofvarious histone H3 cDNAs (pH3c-l; pH3c-ll) by Wu etal.(1989) has made possible more comprehensive studies ontranscript levels of various H3 histone genes and the roleof histone variants in cultured plants cells. Northernanalysis with 3'-end-specific probes has showed anS-phase-specific expression for the H3c-1 gene and areplication-independent expression for H3c-ll. The ex-pression of a later histone H3 variant was influenced byseveral external stress factors and its transcripts wereaccumulated in the early stage of somatic embryodevelopment with higher abundance (Kapros et al. 1991).The possible functional significance of higher amounts oftranscripts from the H3c-ll variant gene during early

Embryogenesis from somatic plant cells 477

X1CT3

6 6 —

45—"

36 —

29—t24— *

MSPRP1

MsCal

SbPRPI

366—

Msc27

1 2 3 4 5 6 7 8

45—

36—

29 —24 —

1 2 5 6 7 8 9 10 11 12

Fig. 4. Changes in p34cdc2 kinase activity in protoplast-derivedalfalfa cells during in vitro culture. (A) Silver staining ofproteins bound to plS^-Sepharose after SDS-PAGE.(B) Autoradiography of the fractions in A after in vitrophosphorylation assay in the presence of histone HI. Time ofsampling during culture, 1, 0; 2, 6h; 3, 12 h; 4, 24 h; 5, 36 h; 6,48 h; 7, 60 h; 8, 72 h; 9, 4 days; 10, 5 days; 11, 6 days; 12, 7days (L. Bak6, unpublished).

embryogenesis might be suggested by the finding that thishistone H3 variant showed a high steady-state level ofacetylation in alfalfa (Waterborg et al. 1989). In view of thesuggestion about the role of histone acetylation in theformation of transcriptionally active chromatin (Megee etal. 1990) or about the signal function of acetylation, whichmodulates histone-protein and histone-DNA interaction(Loidl, 1988), it can be proposed that this histone variant isan active component in the alteration of nucleosomestructure. Activation of a set of genes with a coordinatedexpression pattern might occur during induction of theembryogenic pathway through reactivation of the cellcycle.

Early and late marker genes with alteredexpression during the inductive phase and theprogression of somatic embryogenesis

If the developmental switch in somatic cells at the time ofembryogenic induction involves an overall reprogram-

478 D. Dudits et al.

Fig. 5. Expression of auxin-responsive genes in 2,4-D-treatedmicrocallus suspensions (MCS) and in alfalfa somatic embryos.MsPRPl, alfalfa cDNA encodes for a proline-nch protein(unpublished); MsCal, alfalfa cDNA encodes for a putativeCa*+-binding protein (K. N6meth, unpublished); SbPRPI,soybean cDNA encodes a proline-rich protein (Hong et al.1987); Msc27, alfalfa cDNA used as a reference. For Northernanalysis total RNA was isolated from MSC after a 1 htreatment with 100/iM 2,4-D (lane 1) and subsequent culturein hormone-free medium for l h (2); 3h (3); 8h (4); 1 day (5); 3days (6); 7 days (7) and 21 days (8). Ei, early stage of globularembryos; E2, heart-stage embryos; E3, elongated torpedo-shaped embryos.

ming of gene expression as a consequence of externalstimuli mediated by signal transducers, one should be ableto identify key genes in these complex molecular andcellular processes. Cloning of genes with differentialexpression during the onset of the embryogenic programor upon completion of embryo development can provideessential tools for further comprehensive analysis of theunderlying molecular events. These studies on carrotsomatic embryogenesis have been initiated in the earlyeighties, as reviewed by Nomura and Komamine (1986).

Detection of so-called early marker genes expresseddifferentially in somatic cells during the phase ofcommitment towards embryogenic development and for-mation of a totipotent state requires the use of tissueculture systems based on primary explants or protoplastsfrom plant organs. Alternatively, continuously main-tained suspension culture can be used if the exact timingof the induction phase in embryo formation is possible.Differential screening of a cDNA library constructed frompoly(A)+ RNA of 2,4-D-treated microcallus suspension(MCS) of alfalfa (shown in Fig. 1A) has revealed a set of2,4-D-responsive genes with characteristic expressionpatterns during the transition to embryogenesis and thesubsequent stages of embryo development (Fig. 5). TheMsPRPl cDNA encodes a proline-rich, putative cell wallprotein (J. Gyo'rgyey, unpublished). This alfalfa geneshows maximum expression at 1-3 days after 2,4-Dtreatment and its transcript cannot be detected in somatic

embryos. In contrast, an auxin-regulated gene encodinganother proline-rich protein in soybean (SbPRPl; Hong etal. 1987) exhibits an earlier response and its mRNA isfound in alfalfa somatic embryos in increasing amountsduring later stage of embryogenesis. In soybean theSbPRPl gene is developmentally regulated and highlyexpressed in root, mature hypocotyl and in the early stageof seed coat development (Hong et al. 1989). A number ofrecent studies using various embryogenic culture systemshave shown that 2,4-D treatment, hormone-free growthand wounding can all significantly alter the expression ofvarious proline- or glycine-rich genes (Aleith and Richter,1990; Ludevid et al. 1990). Northern analysis with thesecDNAs suggests a functional role for various cell wallproteins (reviewed by Cassab and Varner, 1988) inhormone-induced cell division and subsequently duringembryo development. Auxin-regulated genes can showembryo-specific expression. As an example, Fig. 5 presentsthe pattern of expression of a putative Ca2+-bindingprotein (MsCal). The 2,4-D treatment of microcallussuspension causes a transient increase in the amount ofMsCal transcripts during hormone-free growth. This geneis preferentially expressed in early globular stage alfalfasomatic embryos (K. N6meth, unpublished result).

In addition to the use of auxin-responsive genes asmolecular markers, the studies on carrot cell suspensioncultures have also resulted in identification of embryo-specific genes and proteins. The comparative analysis ofdifferences between carrot tissues grown in the presence of2,4-D as so-called 'proembryogenic mass' (PEM) andsomatic embryos developed after removal of 2,4-D haveclearly indicated only a limited number of embryo-specificpolypeptides (Sung and Okimoto, 1981; Choi and Sung,1984) and a general similarity in mRNA populations fromPEMs and torpedo-stage embryos (Wilde et al. 1988). Outof several cDNA recombinants representing genes that aredifferentially regulated during carrot somatic embryogen-esis, clone Dc3 is specific for PEM and somatic embryos. Ithas been suggested to be a marker of embryogenicpotential (Wilde et al. 1988; de Vries et al. 1988a). Anothercarrot cDNA clone (DC8) identified by Borkird et al. (1988)detects both somatic and zygotic embryo-specific mRNAspecies with increased amounts in heart-stage somaticembryos (Franz et al. 1989). Antibodies against embryo-specific polypeptide antigens have been produced usingcarrot (Choi et al. 1987) and pea (Stirn and Jacobsen, 1990)cultures.

As a characteristic feature of carrot cultures it wasfound that a set of extracellular proteins secreted into theculture medium could promote the acquisition of embryo-genic potential (De Vries et al. 1988a,b). cDNA clonescorresponding to these extracellular proteins have alsobeen identified. On the basis of Northern analysis, one ofthese genes (PS48) is expressed only in embryogenic lines(Booij et al. 1990).

Despite the fact that several of the above mentionedmolecular markers are available at present, essentialinformation is still missing to enable us to understand thebasic molecular processes involved in the transition fromsomatic to embryogenic cell type. A working hypothesiscould be proposed that the formation of embryos in vitro isa stress response. During establishment of the cultures thesomatic cells or tissues are removed from their originalenvironment and exposed to different stresses includingartificial growth conditions, in vitro starvation, superopti-mal hormone concentration and wounding. Generation ofan embryogenic state in somatic cells could be part of a

general adaptation process. In this case cellular functionslinked to the stress response can play a role in activation ofthe embryogenic developmental program. Involvement ofthe heat-shock system is supported by a number ofexperimental results. An altered heat-shock response wasobserved in carrot cell suspension cultures during theformation of somatic embryos (Pitto et al. 1983; Zimmer-mann et al. 1989). Heat-shock genes can be activated by2,4-D treatment in soybean hypocotyl tissues (Czarneckaet al. 1984). A temperature-sensitive non-embryogeniccarrot mutant (ts 59) is defective in phosphorylation ofheat-shock proteins (see Terzi and Lo Schiavo, 1990). Inagreement with reports on transient heat-shock geneexpression in early stages of animal embryogenesis(reviewed by Bond and Schlesinger, 1987; Lindquist andCraig, 1988) a small heat-shock gene (Mshspl8) was foundto be expressed in early stages of alfalfa somatic embryosunder normal culture conditions (Gydrgyey et al. 1991).This heat-shock protein contains the GVLTV amino acidmotif that is characteristic of proteins with aggregationcapability (Ingolia and Craig, 1982). On the basis ofpresent knowledge about molecular chaperones (Ellis,1990) heat-shock proteins may have this function toensure proper folding or assembly of cellular proteinsduring a change in the developmental program such asinitiation of somatic embryogenesis.

Considering the morphological similarities betweenzygotic and somatic embryos during progression of embryodevelopment, one would expect the same genes to be activein the various developmental stages in both cases. The roleof abscisic acid (ABA) in embryo maturation and seeddevelopment by regulation of gene expression has beenproved by studies on a number of zygotic embryos(reviewed by Quatrano, 1986). Exogenously applied ABAcan significantly improve the somatic embryogenesis invitro (Ammirato, 1977). The expression of the DC8embryogenic gene of carrot is dependent on ABA (Sung etal. 1990). Embryo-specific gene expression in somaticembryos is supposed to be under tight developmentalcontrol, as demonstrated for zygotic embryogenesis.Regulation of gene expression during plant embryogenesishas been recently reviewed by Goldberg et al. (1989).

Global reorganization of cyto-architecture:polarization during induction of somatic embryodevelopment

As a result of auxin treatment there is a considerablechange in cell morphology that reflects the reorganizationof structural components of somatic cells. Both the cell sizeand the type of first division are distinctly differentbetween cells committed to embryogenesis and cells thatform callus tissue (see Fig. 3). The embryogenic cells aresmall and highly cytoplasmic. Asymmetric division canfrequently be observed in cultures induced to startembryogenesis. The greatest difference in microtubuleorganization between embryogenic and non-embryogeniccells can be seen during the second day of alfalfa protoplastcultures (Dijak and Simmonds, 1988). In non-induced cellsthe microtubules were organized into thick, parallelbundles with high a degree of order. The induced cellspossessed fine, more numerous microtubular strandsarranged in a disordered network. During initiation ofembryogenesis in carrot PEM the number of corticalmicrotubules increases (Cyr et al. 1987). In hypocotyl

Embryogenesis from somatic plant cells 479

tissues auxin, like indolebutyric acid, increases thesynthesis of tubulin (Kantharaj et al. 1985).

Asymmetry in the first division may be a prerequisitefor further polar growth during embryogenesis. Theinvolvement of actin filaments in nuclear positioning hasbeen suggested by studies on tobacco cells from protoplasts(Katsuta and Shibaoka, 1988). During Fucus embryogen-esis actin microfilaments play a crucial role in theestablishment of an embryogenic axis (Kropf et al. 1989).The involvement of actin in cellular processes such as cellshape determination and karyokinesis has been discussedby Staiger and Schliwa (1987). At present no regulatorymechanism is known that could be responsible for thespatial distribution of cells with distinct characteristicsduring the subsequent cell divisions throughout embryo-genic stages (globular, heart, torpedo) and, finally, theformation of root and shoot meristem.

Perspectives

Out of the few plant-specific cellular functions theplasticity of'the differentiated cell stage' may be of centralimportance for developmental molecular biology. Somaticplant cells offer a unique experimental system forunderstanding the key components in the developmentalswitch that is induced by hormones through reactivationor alteration of the cell cycle, which influences cellmorphology, the characteristics of cell division and theinitiation of polarized growth. To date, studies on somaticembryo formation have been dominated by tissue cultureexperiments. The use of biochemical and molecularapproaches has just started in recent years. Furtherprogress in understanding somatic embryogenesis as adevelopmental pathway will be highly dependent onimprovement of culture systems, on extending the use ofdevelopmental mutants and on isolation and characteriz-ation of genes involved in hormone-mediated cellularfunctions. Recognition of the regulatory mechanisms thatare responsible for coordinated reprogramming of geneexpression patterns will require extensive molecularcloning work complemented by functional analysis usingtransgenic plants. Without an understanding of themolecular basis of the mode of hormone action, studies onsomatic embryogenesis will remain purely empirical.Characterization of the components involved in signallingin hormone-treated cells and isolation of cell cycle geneswill result in significant progress in research intoembryogenesis. There is also the need to find the linksbetween altered gene expression and structural changes individing cells and developing embryos.

The authors thank Professor Jakob H. Waterborg for criticalreading of the manuscript and Bela Dusha for the excellentphotographic work.

References

ABDULLAH, R., COCKING, E. C. AND THOMPSON, J. A. (1986). Efficientplant regeneration from rice protoplasts through somaticembryogenesis. Bio/Technol 4, 1087-1090.

ALEITH, F. AND RICHTER, G. (1990). Gene expression during induction ofsomatic embryogenesis in carrot cell suspensions. Planta 183, 17-24.

AMMIRATO, P. V. (1977). Hormonal control of somatic embryodevelopment from cultured cells of caraway. PL Physwl. 59, 579-586.

AMMIBATO, P. V. (1983). Embryogenesis. In Handbook of Plant CellCulture (ed. D. A. Evans, W. R. Sharp, P. V. Ammirato, Y. Yamada),vol. 1., pp. 82-123. New York: Macmillan Publishing Co.

BAKO, L. BOGRE, L. AND DuDrrs, D. (1991). Protein phosphorylation in

partially synchronized cell suspension culture of alfalfa, hi NATOAdv. Studies on Cellular Regulation by Protein Phosphorylation (ed L.Heilmayer), (in press).

BARBIER-BRYGOO, H., EPHRITIKHINE, G., KLAMBT, D., GHISLAIN, M. ANDGUERN, J. (1989). Functional evidence for an auxin receptor at theplasmalemma of tobacco mesophyll protoplasts. Proc. natn. Acad. Sci.U.SA 86,891-895.

BARBIBR-BYRGOO, H., GUERN, J., EPHIRITIKHINE, G., SHEN, W. H.,MAUREL, C. AND KLAMBT, D. (1990). The sensitivity of plantprotoplats to auxins: modulation of receptors at the plasmalemma. InPlant Gene Transfer (ed. C. Lamb and R. Beachy) New York: Loss (inpress).

BLOW, J. (1989). Mitosis comes apart. Trends Genet. 5, 166-167.BLUM, W., HINSOH, K. D., SCHULTZ, G. AND WBILER, E. W. (1988).

Identification of GTP-binding proteins in the plasma membrane ofhigher plants. Biochem. biophys. Res. Commun. 156, 954-959.

BOGRE, L., OLAH, Z. AND DUDITS, D. (1988). Ca2+-dependent proteinkinase from alfalfa (Medicago varia). partial purification andautophosphorylation. Plant. Sci. 58, 135-144.

BOGRE, L., STEFANOV, I., ABRAHAM, M., SOMOGYI, I. AND DUDITS, D.(1990). Differences in responses to 2,4-dichlorophenoxyacetic acid (2,4-D) treatment between embryogenic and non-embryogenic lines ofalfalfa. In Progress in Plant Cellular and Molecular Biology (ed. H. J.J. Nijkamp, L. H. W. van der Plas and J. van Aartrijk), pp. 427-436.Dordrecht Kluwer Acad. Publ.

BOND, U AND SCHLESINGER, M. J. (1987). Heat-shock proteins. A. Rev.Genet. 22, 631-677.

Boou, H., STERK, P., SCHELLKKENS, G. A., VAN KAMMBN, A. AND DEVRIES, S. C. (1990). Tissue and cell-specific expression of genesencoding carrot extracellular proteins. In Progress in Plant Cellularand Molecular Biology (ed. H. J. J. Nijkamp, L. H. W. van der Plasand J. van Aartrijk), pp. 398—401, Dordrecht: Kluwer Acad. Publ.

BORKIRD, C, CHOI, J. H., JIN, Z. H., FRANZ, G., HATZOPOULOS, P ,CHORNEAU, R., BONAS, U., PELBGRJ, F. AND SUNG, Z R. (1988).Developmental regulation of embryogenic genes in plants. Proc. natn.Acad. Sci. U.SA. 85, 6399-6403.

BRIZUELA, L., DRAETTA, G. AND BEACH, D. (1987). P13"*1 acts in thefission yeast cell division cycle as a component of the p34cdc2 proteinkinase. EMBO J. 6, 3507-3514.

BROWN, D. C. W. AND ATANASSOV, A. (1985). Role of genetic backgroundin somatic embryogenesis in Medicago. PI. Cell Tiss. Organ Culture 4,111-122.

BRUMMBL, D. A. AND HALL, J. L. (1987). Rapid cellular responses toauxin and the regulation of growth. Plant, Cell Environ. 10, 523-543.

CASSAB, G I. AND VARNER, J. E. (1988) Cell wall proteins. A Rev. PI.Physwl. PI. molec. Biol. 39, 321-353.

CHOI, J. H., LIN, L. S., BORKIRD, C. AND SUNQ, Z. R. (1987). Cloning ofgenes developmentally regulated during plant embryogenesis. Proc.natn. Acad Sci. U.SA. 84, 1906-1910.

CHOI, J. H. AND SUNG, Z. R. (1984). Two-dimensional gel analysis ofcarrot somatic embryogenic proteins. PI. molec. Biol. Rep. 2, 19-25.

CYR, R. J., BUSTOS, M. M., GUILTINAN, M. J. AND FOSKET, D. E. (1987).Developmental modulation of tubulin protein and mRNA levelsduring somatic embryogenesis in cultured carrot cells. Planta 171,365-376.

CZARNECKA, E., EDELMAN, L., SCHOFFL, F. AND KEY, J. L. (1984).Comparative analysis of physical stress responses in soybeanseedlingB using cloned heat shock cDNAs. PL molec. Biol. 3, 45-58.

DAVIES, E. (1987). Action potentials as multifunctional signals in plants:a unifying hypothesis to explain apparently disparate woundresponses. PL Cell Environ. 10, 623-631.

DE VRIES, S C, BOOU, H., JANSSENS, R., VOGELS, R., SARIS, L., LOSCHIAVO, F., TERZI, M. AND VAN KAMMEN, A. (19886). Carrot somaticembryogenesis depends on the phytohormone-controlled presence ofcorrectly glycosylated extracellular proteins. Genes Dev. 2, 462-476.

DE VRIES, S C, BOOU, H., MEYERINK, P., HUISMAN, G., WILDE, H. D.,THOMAS, T. L. AND VAN KAMMEN, A. (1988a). Acquisition ofembryogenic potential in carrot cell-suspension cultures. Planta 176,196-204.

DIJAK, M. AND SIMMONDS, D. H. (1988). Microtubule organization duringearly direct embryogenesis from mesophyll protoplasts of Medicagosativa. PL Sci. 58, 183-191.

DUAK, M., SMITH, D. L., WILSON, T. J. AND BROWN, D. C. W. (1986).Stimulation of direct embryogenesis from mesophyll protoplasts ofMedicago sativa. PL Cell Rep. 5, 468-470.

DBAETTA, G. (1990). Cell cycle control in eukaryotes: molecularmechanisms of cdc2 activation. Trends biochem. Sci. 15, 378—383.

DUDITS, D. (1987). New approaches to genetic manipulation of plants. InCell Culture and Somatic Cell Genetics of Plants (ed. F. Constabel andI. K. Vasil), vol. 4, pp. 139-152. New York: Academic Press.

ELLIS, R. J. (1990). Molecular chaperones: the plant connection. Science250, 954-959.

480 D. Dudits et al.

EPBL, D. (1990). The initiation of development at fertilization. CellDiffer. Dev. 29, 1-12.

ETTLINGER, C. AND LEHLE, L. (1988). Auxin induces rapid changes inphosphatidylinositol metabolites. Nature 331, 176-178.

FEILER, H. S. AND JACOBS, T. W. (1990). Cell division in higher plants- Acdc2 gene, its 34-kDa product, and histone HI kinase activity in pea.Proc. natn. Acad. Sci. U.S.A. 87, 5397-5401.

FRANZ, G., HATZOPOULOS, P., JONES, T. J., KRAUSS, M. AND SUNG, Z. R.(1989). Molecular and genetic analysis of an embryogenic gene, DC8,from Daucus carota L. Molec. gen. Genet. 218, 143-151

FUJIMURA, T. AND KOMAMINE, A. (1980). The serial observation ofembryogenesis in a carrot cell suspension culture. New Phytol. 86,213-218.

FURUKAWA, Y., PIWNICA-WORMS, H., ERNST, T. J., KANAKURA, Y. ANDGRIFFIN, I. D. (1990). cdc2 gene expression at the Gl to S transition inhuman T lymphocytes. Science 260, 805-808.

GIORDANO, A., WHYTE, P., HARLOW, E., FRANZA, R. B., BEACH, JR D. ANDDRABTTA, G. (1989). A 60 kD cdc2-associated polypeptide complexeswith the E1A proteins in adenovinis-infected cells. Cell 58, 981-990.

GOLDBERG, R. B., BARKER, S. J. AND PEREZ-GRAN, L. (1989). Regulationof gene expression during plant embryogenesis. Cell 56, 149-160.

GUERN, J., EPHRITIKHINE, G., IMHOFT, V. AND PRADIER, J. M. (1990).Signal transduction at the membrane level of plant cells. In Progressm Plant Cellular and Molecular Biology (ed. H. J. J Nijkamp, L. HW. van der Plas and J. van Aartrijk1), pp. 466-479. Dordrecht: KluwerAcad. Publ.

GYORGYBY, J., GARTNER, A., NEMETH, K., MAGYAR, Z., HIRT, H.,HKBERLE-BORS, E AND DUDITS, D. (1991). Alfalfa heat-shock genes aredifferentially expressed during somatic embryogenesis. PI. molec. Biol.(in press).

HAHMON, A. C, PUTMAN-EVANS, C. AND CORMIER, M. J. (1987). Acalcium dependent but calmodulin-independent protein kinase fromsoybean. PI. Physiol. 83, 830-837.

HESSE, T, FELDWISCH, J., BALSHUSEMANN, D., BAUW, G., PUYPE, M.,VANDBKERKCHOVE, J., LOBLEH, M., KLAMBT, D., SCHELL, J. AND PALME,K. (1989). Molecular cloning and structural analysis of a gene fromZea mays (L.) coding for a putative receptor for the plant hormoneauxin. EMBO J. 8, 2453-2461.

HIRT, H., PAY, A., GYORGYEY, J., BAKO, L., NEMETH, K., BOORE, L.,SCHWBYKN, R. J , HlBERLE-BOBfl, E AND DUDITS, D. (1991).Complementation of a yeast cell cycle mutant by an alfalfa cDNAencoding a protein kinase homologous to 034™*". Proc. natn. Acad.Sci. U.S.A. 88, 1636-1640.

HONG, J. C, NAGAO, R. T. AND KEY, J. L. (1987). Characterization andsequence analysis of a developmentally regulated putative cell wallprotein gene isolated from soybean. J. bwl. Chem. 262, 8367-8376.

HONG, J. C, NAOAO, R. T. AND KEY, J. L. (1989). Developmentallyregulated expression of soybean proline-rich cell wall protein geneThe PI. Cell 1, 937-943.

HUNT, T. (1989). Maturation promoting factor, cyclin and control of M-phaae. Current Opinion Cell Biol. 1, 268-274.

INGOLJA, T. D. AND CRAIG, E. A. (1982). Four small DroBophila heatshock proteins are related to each other and to mammalian a-crystallin. Proc. natn. Acad. Sci. U.S.A. 79, 2360-2364

INOHARA, N., SHIMOMURA, S., FUKUI, T. AND FUTAI, M. (1989). Auxin-binding protein located in the endoplasmic reticulum of maize shoots-molecular cloning and complete primary structure. Proc. natn. Acad.Sci. U.S.A. 86, 3564-3568

JOHN, P. C. 1 ,̂ SBK, F. J., CARMICHAEL, J. P. AND MCCURDY, D. W.(1990). p34c"2 homologue level, cell division, phytohormoneresponsiveness and cell differentiation in wheat leaves. J Cell Sci. 97,627-630.

JOHN, P. C. L., SKK, F. J. AND LEB, M. G. (1989). A homolog of the cellcycle control protein p34cdc2 participates in the division cycle ofChlamydomonas, and a similar protein is detectable in higher plantsand remote taxa. PI. Cell 1, 1185-1193.

KANTHARAJ, G. R , MAHADEVAN, S. AND PADMANABHAN, G. (1985).Tubulin synthesis and auxin-induced root initiation in Phaseolus.Phytochemistry 24, 23-37.

KAO, K. N AND MICHAYLUK, M H. (1980). Plant regeneration frommesophyll protoplasts of alfalfa. 2. Pflanzenphysiol. 96, 135-141.

KAPROS, T., BOGBE, L., NEMETH, K., BAK6, L., GYOROYEY, J., Wu, S. CAND DUDITS, D. (1991). Differential expression of histone H3 genevariants during cell cycle and somatic embryogenesis in alfalfa,(submitted)

KATSUTA, J. AND SHIBAOKA, H. (1988). The roles of the cytoskeleton andcell wall in nuclear positioning in tobacco by BY-2 cells. Plant CellPhysiol. 29, 403-413.

KELLY, G. J. (1984). Calcium, calmodulin and the action of planthormones. Trends Biochem Sci. 9, 4-5.

KLAMBT, D. (1990). A view about the function of auxin-binding proteinsat plasma membranes. PI. molec. Biol. 14, 1045-1050.

KLUCIS, E. AND POLYA, G. M. (1988). Localization, solubilization and

characterization of plant membrane-associated calcium-dependentprotein kinases. PI. Physiol. 88, 164-171.

KOMAMINE, A., MATSUMOTO, M., TSUKAHARA, M., FUJIWARA, A ,KAWAHARA, R., ITO, M., SMITH, J., NOMURA, K. AND FUJIMURA, T.(1990). Mechanisms of somatic embryogenesis in cell cultures -physiology, biochemistry and molecular biology In Progress m PlantCellular and Molecular Biology (ed. H. J. J. Nrjkamp, L. H. W. vander Plas and J. van Aartrijk), pp 307-313. Dordrecht: Kluwer Acad.Publ.

KROPF, D L., BERGE, S. K. AND QATRANO, R. S. (1989). Actin localizationduring Fucus embryogenesis The Plant Cell 1, 191-200.

LINDQUIST AND CRAIG (1988). The heat-shock proteins. A. Rev Genet. 22,631-677

LOIDL, P. (1988). Towards an understanding of the biological function ofhistone acetylation. FEBS Lett. 227, 91-96.

LUDEVID, M. D., RUIZ-AVILA, L., VALUES, M. P., STIEFEL, V., TORRENT,M., TORNE, J M. AND PUIGDOMENECH, P (1990). Expression of gene3for cell wall proteins in dividing and wounded tissues of Zea mays LPlanta 180, 524-529.

MCVEY, D., BRIZUELA, L., MOHR, I., MARSHALZ, D. R., GLUZMAN, Y. ANDBEACH, D. (1989). Phosphorylation of large tumor antigen by cdc2stimulates SV40 DNA replication. Nature 341, 503-507.

MEGEE, P. C, MORGAN, B. A., MITTMAN, B. A. AND SMITH, M. M. (1990).Genetic analysis of histone H4: essential role of lysines subject toreversible acetylation. Science 247, 841-845.

MORENO, S AND NURSE, P. (1990). Substrates for p34cdca in uivo veritasCell 61, 649-561.

MOROCZ, S., DONN, G., NEMETH, J. AND DUDITS, D (1990). An improvedsystem to obtain fertile regenerants via maize protoplasts isolatedfrom a highly embryogenic suspension culture. Theor. appl. Genet 80,721-726.

MORRE, D. J., GRIPSHOVER, B., MONROE, A. AND MORRE, J. T. (1984).Phosphatidylinositol turnover in isolated soybean membranesstimulated by the synthetic growth hormone 2,4-dichlorophenoxyacetic acid. J. biol. Chem. 259, 15 364-15 368.

MORSE, M. J., SATTER, R. L., CRAIN, R. C. AND COTE, G. G. (1989). Signaltransduction and phosphatidylinositol turnover in plants. PhysiologiaPI. 76, 118-121.

MURRAY, A W AND KIRSCHNER, M W. (1989). Cyclin synthesis drivesthe early embryogenic cell cycle Nature 338, 275-280.

NOMURA, K. AND KOMAMINE, A. (1985). Identification and isolation ofsingle cells that produce somatic embryos at a high frequency in acarrot suspension culture. PI. Physiol 79, 988-991.

NOMURA, K. AND KOMAMINE, A. (1986). Molecular mechanisms ofsomatic embryogenesis. Oxford Surveys of Plant Molecular and CellBiology, vol. 3, pp. 467-466.

OLAH, Z., BOGRE, L., LEHEL, CS., FARAGO, A , SEPHODI, J. AND DUDITS, D(1989). The phosphorylation site of Ca2+-dependent protein kinasefrom alfalfa. PI molec. Bwl. 12, 453-461.

OLD, R. W. AND WOODLAND, H. R. (1984). Histone genes- not so simpleafter all. Cell 38, 624-626

PIT-TO, L., Lo SCHIAVO, F., GruuANO, G AND TERZI, M. (1983). Analysisof the heat shock protein pattern during somatic embryogenesis ofcarrot PI. molec. Biol. 2, 231-237.

POOVAIAH, B. W , REDDY, A. S. N. AND MCFADDEN, J. J. (1987) Calciummessenger system: role of protein phosphorylation and inositol andbiphospholipids Physiologia PI. 69, 669-573.

QUATRANO, R. S. (1986). Regulation of gene expression by abscisic acidduring angiosperm embryo development. Oxford Surveys of PlantMolecular and Cell Biology, vol 2, pp. 467-477.

RACUSEN, R. H. AND SCHIAVONE, F. M (1990). Positional cues anddifferential gene expression in somatic embryos of higher plants. CellDiffer. Dev. 30, 159-169.

RANJEVA, R., CARRASCO, A. AND BOUDET, A. M. (1988). Inositoltriphosphate stimulates the release of calcium from intact vacuolesisolated from Acer cell. FEBS Lett. 230, 137-141.

REINERT, J. (1969). tJber die Kontrolle der Morphogenese und dieInduktion von Adventivembryoone an Gewebekulturen aus Karrotten.Planta 53, 318-333.

SCHUMAKER, K. S. AND SZE, H (1987). Inositol 1,4,5,-triphosphatereleases Ca2+ from vascular membrane vesicles of oat roots. J. biol.Chem. 262, 3944-3946

SCHUMPERU, D. (1988). Multilevel regulation of replication-dependenthistone genes. Trends Genet. 4, 187-191.

SHEN, W. H., PETIT, A., GUERN, J AND TEMPE, J. (1988). Hairy roots aremore sensitive to auxin than normal roots. Proc. natn. Acad. Sci.U.S.A. 88, 3417-3421.

SHIMOMURA, S., INOHARA, N., FUKUI, T. AND FUTAI, M. (1988). Differentproperties of two types of auxin-binding sites in membranes frommaize coleoptiles Planta 175, 588-566.

SMITH, D. L. AND KRIKORIAN, A. D. (1990). pH control of carrot somaticembryogenesis. In Progress in Plant Cellular and Molecular Biology

Embryogenesis from somatic plant cells 481

(ed. H. J. J. Nijkamp, L. H. W. van der Plas, and J. van Aartnjk), pp.449-453. Dordrecht: Kluwer Acad. Publ

SOLOMON, M. J., GLOTZER, M., LEE, T. H., PHILIPPE, M AND KIRSCHNER,M. (1990). Cyclin activation of p34cdc3. Cell 63, 1013-1024.

SONG, J., SORENSEN, E. L. AND LIANG, G. H (1990) Directembryogenesia from single mesophyll protoplasts in alfalfa (Medicagosatiua L.). PI Cell Rep. 9, 21-25.

SPEKSNUDER, J. A , CORSON, D. W., SARDET, C. AND JAFFE, L. F. (1989).Free calcium pulses following fertilization in the ascidian egg. DeviBiol. 135, 182-190.

STAIGBR, C. J. AND SCHLJWA, M (1987). Actin localization and functionin higher plants. Protoplasma 141, 1-12.

STEWARDS, F. C, MAPBS, M O AND MEARS, K. (1958). Growth andorganized development of cultured cells. II. Organization in culturesgrown from freely suspended cells Am. J. Bot. 45, 705-708.

STIRN, S. AND JACOBSBN, H. J. (1990). Production and characterization ofmonoclonal antibodies against marker proteins for somaticembryogenesis in pea (Pistum satwum L.). In Progress in PlantCellular and Molecular Biology (ed H. J J Nijkamp, L. H. W. vander Plas and J. van Aartrijk), pp. 460—465, Dordrecht: Kluwer Acad.Publ

SUNG, Z. R., FRANZ, G., HATZOPOULOS, P., GOUPIL, P. AND HEMPEL, F.(1990) How do leaf cells become embryogenic cells? Abstract EMBOWorkshop on Molecular Basis of Plant Embryogenesis, Wageningen,Agricultural University.

SUNG, Z. R. AND OKIMOTO, R. (1981). Embryogenic proteins in somaticembryos of carrot. Proc. natn. Acad Sci. U.S A. 78, 3683-3687.

TERZI, M AND LO SCHIAVO, F. (1990). Developmental mutants in carrot.In Progress in Plant Cellular and Molecular Biology (ed. H. J. J.Nykarop, L. H. W van der Plas and J. van Aartrijk), pp. 391-397.Dordrecht. Kluwer Acad. Publ.

TILMANN, U., KAYSER, V. G., SLEMEIOTER, G., HESSE, T., PALME, K.,LOBLBR, M. AND KLAMBT, D. (1989) cDNA clones of the auxin-bindingprotein from corn coleoptiles (Zea mays L.) Isolation andcharacterization by immunological methods. EMBO J. 8, 2463-2467.

TIMMERS, A C J., DE VRTBS, S. C. AND SCHKL, J. H. N. (1989).

Distribution of membrane-bound calcium and activated calmodulinduring somatic embryogenesis of carrot (Daucus carota L.).Protoplasma 153, 24-29.

VASIL, V , REDWAY, F. AND VASIL, I. K. (1990). Regeneration of plantsfrom embryogenic suspension culture protoplasts of wheat (Triticumaestwum L.). Bio/Technol. 8, 429-434.

WARREN, G. S. AND FOWLER, M. W. (1978). Cell number and celldoubling times during the development of carrot embryoids insuspension culture. Experimentia 34, 356-357.

WATERBORG, J. H., HARRINGTON, R. E. AND WINICOV, J. (1989).Differential histone acetylation in alfalfa (Medicago sativa) due togrowth in NaCl. PI. Physiol. 90, 237-245.

WATERBORG, J. H., WINICOV, I. AND HARRINGTON, R. E. (1987). Histonevariants and acetylated species from the alfalfa plant Medicago satwa.Archs Biochem. Biophys. 256, 167-178.

WERNICKE, W. AND MILKOVITS, L (1987). Effect of auxin on mitotic cellcycle in cultured leaf segments at different stages of development inwheat. Physiologia PI. 69, 16-22.

WERNICKE, W., ROS, M. AND JUNG, G. (1990). Microtubules and the firstcell cycle in cultured mesophyll protoplasts of Nicotiana. In Progressin Plant Cellular and Molecular Biology (ed. H. J. J. Nijkamp, L. H.W. van der Plas and J. van Aartrijk), pp 538-543. Dordrecht: KluwerAcad. Publ.

WILDE, H. D., NELSON, W. S., BOOJI, H., DE VRJES, S. C. AND THOMAS, T.L. (1988). Gene-expression programs in embryonic and non-embryogenic carrot cultures. Planta 176, 205-211.

Wu, S. C, GYORGYBY, J. AND DUDITS, D. (1989) Polyadenylated H3hiatone transcripts and H3 histone variants in alfalfa. Nucl. Acids res.17, 3057-3063.

ZBEIX, B. AND WALTER-BACK, C. (1988) Signal transduction of auxin onisolated plant cell membranes: indications for a rapidpolyphosphoinositide response stimulated by indoleacetic acid. J. PI.Physiol. 133, 353-360.

ZlMMERMANN, J. L., APUYA, N., DARWISH, K. AND O'CARROLL, C. (1989).Novel regulation of heat shock genes during carrot somatic embryodevelopment. The PI. Cell 1, 1137-1146.

482 D. Dudits et al.