Plant Cell Reports (1999) 18: 929-934 0 Springer-Verlag 1999

J.S. Nonohay . J.E.A. Mariath . H. Winge

Histological analysis of somatic embryogenesis in Brazilian cultivarsof barley, Hordeum vulgare vulgare, Poaceae

Received: 4 June 1998 / Revision received: 28 August 1998 / Accepted: 7 December 1998

Abstract Histological analysis was performed aimed atelucidating the origin and the developmental process ofsomatic embryos of two Brazilian cultivars of barley(Hordeum w&are w&are), ‘MN-599’ and ‘A-05’. Theobserved site of somatic embryo origin (SSEO) couldoriginate from a superficial callus cell, possibly indi-cating a unicellular origin, or from epidermal and sube-pidermal callus cells, representing a multicellularorigin. A fold, the somatic embryo scutellum thatsubsequently develops into a cotyledonary leaf, indi-cates the somatic embryo differentiation. The somaticembryos also showed a growth increase of the primaryroot and, occasionally, a delay in root development. Apossible alternative pathway for the origin of somaticembryos is suggested, in which a SSEO forms a clumpof somatic embryos.

Key words Hordeum vulgare vulgare . Barley .Somatic embryogenesis . Histological analysis

Introduction

Somatic embryogenesis was defined by Emons (1994)as the development from somatic cells of structures thatfollow a histodifferentiation pattern which leads to abody pattern resembling that of zygotic embryos. This

Communicated by H. L&-z

J.S. Nonohay . H. Winge (m)Departamento de Genetica, Universidade Federal do RioGrande do Sul, Caixa Postal 15053, CEP 91501-970,Porto Alegre, RS, Brazile-mail: winge@vortex.ufrgs.br or helwinge@if.ufrgs.brFax: +.5.5-51-3192011

J.E.A. MariathDepartamento de Botanica, Universidade Federal do RioGrande do Sul, Caixa Postal 15053, CEP 91501~970.Porto Alegre, RS, Brazil

process occurs naturally in some species of plants andcan be induced during tissue culture of diverse speciesof plants. In vitro, somatic embryogenesis can eitheroccur directly, from cells of an organized tissue, or indi-rectly, from callus or suspension culture (Williams andMaheswaran 1986).

Somatic embryos are organized bipolar structuresarising from a single cell and having no vascularconnection with maternal tissue (Haccius 1978).However, with respect to indirect somatic embryogen-esis there is dissension regarding the uni- or multicel-lular origin of the somatic embryos. According toWilliams and Maheswaran (1986) “there appears to beuniversal formation of a compact clump of embryog-enic cells, the proembryonal complex, from which oneto many embryoids develop”. Histological studies ofsomatic embryos of different species have describedboth pathways of origin: unicellular (Vasil and Vasil1982; McCain et al. 1988; Jones and Rost 1989) andmulticellular (Wernicke et al. 1982; McCain andHodges 1986; Taylor and Vasil 1996).

Another controversial point is the similarity ordissimilarity between the processes of zygotic andsomatic embryogenesis. In barley, the development ofthe somatic embryo is usually atypical when comparedto that of the zygotic embryo (Thomas and Scott 1985).Vasil and Vasil (1982), Botti and Vasil (1984) andTaylor and Vasil (1996) also studied the differencesbetween zygotic and somatic embryos.

Somatic embryogenesis in barley has been reportedin a number of papers, e.g., Thomas and Scott (1985),Ltihrs and Liirz (1987), Ruiz et al. (1992) and King andKasha (1994), although only a limited number of histo-logical reports are available (Mohanty and Ghosh 1988;Ryschka et al. 1991; Oka et al. 1995).

The histological study presented here was designedto analyze the somatic embryogenesis pathway inbarley (Hordeum vulgare ssp vulgare), beginning withthe initial immature embryo explant, in order to eluci-date the origin and development of the resultantembryos.

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Materials and methods

Culture of immature embryos

The two Brazilian cultivars of barley, ‘MN-SYY’ and ‘A-OS used inthis study were obtained from two breeding and brewing compa-nies: C.C. Brahma - Filial Maltaria Navegantes and Cia. Antarc-tica Paulista, respectively. Embryogenic calli, somatic embryos,and plantlets were obtained by culturing immature embryosaccording to the protocols proposed by Ltlhrs and Lorz (1987)and King and Kasha (1994) using MS (Murashige and Skoog1962) and BS long (Carolina Biological Supply, Burlington, N.C.),respectively. as basal media. In the present experiment, theembryonic axis of the immature embryo (0.8-1.5 mm in length)was removed. with only scutellar tissue being used as explantmaterial.

Histological Preparations

The material was fixed by immersion in formalin: acetic acid: SO%ethanol (FAA, 1 : 1: 18 v/v, Johansen 1940) for 48 h and stored in70% ethanol. After dehydration in an ethanol series, the materialwas infiltrated and embedded in hydroxyethylmethacrylate(historesin Jung). Sections 5 km in thickness were obtained with aLeitz 1400 microtome and stained with toluidine blue (0.05% inwater) and hematoxilin. The slides were mounted in CanadaBalsam and analyzed in a Leitz Dialux 20 EB microscope using acamera lucida for schematic representations and Leica MD2camera for photomicrography records.

Results and discussion

Embryogenic calli and somatic embryo formation wereobserved within 2 weeks on both media after cultureinitiation of the two cultivars, as occurred in a formerexperiment (Nonohay 1996). The embryogenic calliwere compact and smooth (Fig. la) and showed smalland dense cells (Fig. 2a) with small vacuoles. Theseembryogenic cells presented anatomical characteristicsthat agree with those described by Williams andMaheswaran (1986) and Emons (1994). According toWilliams and Maheswaran (1986) these are featurescommon to all embryogenic systems.

The removal of the embryonic axis from the imma-ture embryo avoided precocious germination andshowed that the embryogenic calli were formed fromthe scutellum of immature embryos. The embryonicaxis, when cultured, showed precocious germinationand formed only aqueous callus (Nonohay 1996). Otherhistological studies in cereals have also identified thescutellum of immature embryos as being the tissuefrom which the embryogenic calli originate, e.g.,McCain et al. (1988), Ryschka et al. (1991) and Oka etal. (1995).

Our histological data indicated that after severalmitotic divisions, the differentiation of a region withembryogenic cells occurred (Figs. 2c, 3f). This region ofembryogenic cells is assumed to be the proembryonalcomplex, the site of somatic embryo origin (SSEO).Taylor and Vasil (1996) observed the formation of aridge of cells by periclinal and anticlinal divisions in the

Fig. la-f Morphological aspects of barley somatic embryogenes;sfrom immature embryos. a Embryogenic calli from scutcllum(grows). Bar: 1 mm. b Cluster of somatic embryos (arrow). Bar:I mm. c Somatic embryos showing the initial fold (LITTOW). Bur:0.6 mm. d Somatic embryo with a cotyledonary leaf developingfrom an initial fold (arrow). Bar: 1 mm. e Complete regeneratedplantlet showing the primary root (urrow). Bar: 1 mm. f Greenplantlets of barley regenerated by somatic embryogenesis. Bar:2mm

epidermal and subepidermal cells of the scutellum,which marked the beginning of somatic embryo devel-opment. These authors reported that a meristematiczone formed earlier than the ridge as a result of celldivisions that spread throughout the scutellar region.Af te r the format ion of the mer i s temat ic zone ,epidermal cells were nearly identical in size and overallappearance, suggesting that subepidermal cells dividefaster than epidermal cells. This description fitsperfectly in the case under study; we suppose that theformation of the complex of embryogenic cells consti-tutes the morphogenetic sign that initiates somaticembryo differentiation.

An initial periclinal division of a superficial calluscell which, when followed by an anticlinal division, ascan be observed in Figs. 2b, 3a-d, could represent acase of unicellular origin of somatic embryos. Alterna-tively, the SSEO could start by a succession of anti-clinal divisions of the epidermal and subepidermalcontiguous cells, giving rise to a multicellular clump ofembryogenic cells (Fig. 3e). The single- or multicellularorigin of somatic embryos was discussed by Williamsand Maheswaran (1986) who reported that in indirectsomatic embryogenesis the somatic embryos areformed from the proembryonal complex, which couldoriginate from a single cell. On the other hand,according to the authors referred to here, the multicel-lular origin normally produces somatic embryos fusedwith parental tissue, while an unicellular origin givesrise to somatic embryos attached by a suspensor-likeorgan.

Fransz and Schel (1991) proposed the existence of apolarized transitional structure between an embryog-enic unit and the somatic embryo that marks the transi-tion from unorganized to organized growth and deter-mines the initiation of somatic embryogenesis. Thistransitory structure was not observed in our studies butits existence would certainly add an intermediate stagebetween the SSEO and the somatic embryo proper.

The barley somatic embryo, similarly to theembryogenic callus, presented a smooth surface andshowed, as described by Thomas and Scott (1985), afold or lateral notch as a marker of the beginning ofdifferentiation (Fig. lc). As already observed byThomas and Scott (1985) and Mohanthy and Ghosh(1988), we showed histologically that this fold corre-sponds to the scutellum of the somatic embryo(Figs. 2d, 3i) that subsequently developed into a leafystructure (Figs. ld,e, 2e, 2g, 3j,k). We suggest that this

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m ,a b

1

Fig. 3a-k Schematical representation of somatic embryo devel-opment from scutellum of barley immature embryo. a Superficialcallus cell, b first division of a superficial callus cell, c periclinaldivision of a derived superficial callus cell, d anticlinal division ofa derived superficial callus cell, e serial anticlinal divisions ofepidermal contiguous cells, f site of somatic embryo origin(SSEO), g meristematic region without somatic embryo differen-tiation, b group of somatic embryos originated from fused SSEOs,i somatic embryo, j developed somatic embryo, k completelyregenerated plantlet

represents the cotyledon as an overgrowth of thesomatic embryo scutellum (Figs. Ze,f).

Thomas and Scott (1985) reported that the develop-ment of the barley somatic embryo was atypical whencompared to that of zygotic embryos. We observed thedevelopment of the cotyledonary leaf from thescutellum as well as a clear growth increase of theprimary root (Figs. 2g, 2i, 3k), without the formation ofthe first embryonic adventitious root. In the zygoticembryo, this primary root, called coleorhiza, has a shortlifetime since the first adventitious root germinates atthe expense of the original primary root. As far as weknow, this is the first histological presentation for

barley of a somatic embryo having a developed coleo-rhiza.

Occasionally, a delay in root differentiation occursand the plantlet remains connected to the callus, evenwhen the development of the second leaf has alreadyoccurred (Fig. 2j). The late development of rootprimordia in somatic embryos is a common feature inmembers of Poaceae (see Taylor and Vasil 1996).

The presence of the auxin 2,4-dichlorophenoxyaceticacid (2,4-D) in the culture medium may be responsiblefor the dissimilarities between somatic and zygoticembryo development (Vasil and Vasill982) and for theabsence or retardation of shoot-root axis formation(Fransz and Schel 1991).

After root development, a complete plantlet wasregenerated (Fig. le) with cotyledonary leaf, coleoptile,shoot, and root apex and root cap (Figs. 2g-i, 3k).These plants turned green when cultured under light(Fig. lf).

Our histological observations not only enabled us topropose the above suggested pathway for somaticembryogenesis in barley but also disclosed a possiblealternative way. According to this alternative route,somatic embryo development could occur through anintermediary stage in which the meristematic regiongrows without embryo differentiation (Fig. 3g) or anunknown number of SSEO could fuse and then differ-entiate, forming an aggregate of embryos with indepen-dent vascular systems (Figs. lb, 3h). According toRyschka et al. (1991) the embryoid could originatefrom each of the three basic tissues of the scutellum:epidermis, subep idermal l ayer o r p rocambium.Although we have no observations related to the originof somatic embryo from procambium, it is tempting tosuppose that our alternative pathway could originatefrom that tissue.

Acknowledgements We are indebted to Prof. Dr. Horst Liirz,University of Hamburg, Germany, for his critical reading of themanuscript, to Silvia N.C. Richter and Bibiana Cassol for labora-tory assistance and Rinaldo P. dos Santos for the drawings. Thiswork is part of a coordinated program supported by FINEP,FAPERGS, FBB, Accord UFRGS/Cia. Cervejaria Brahma -Maltaria Navegantes, CNPq and CAPES.

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