morphohistological analysis and histochemistry of feijoa sellowiana

Summary. Morphohistological analysis and histochemical studies werecarried out during the induction and development of Feijoa sellowianasomatic embryos. Zygotic embryos were cultured on LPm medium con-taining 2,4-dichlorophenoxyacetic acid (20 �M) and glutamine (8 mM).Somatic embryogenesis could be induced from embryogenic cells thatoriginated in meristematic centers or from clusters of cells. The presenceof few starch grains and abundant protein bodies was observed in theglobular and early torpedo stages, while in torpedo and cotyledonary-stage somatic embryos an enhanced synthesis of starch grains was associ-ated with the accumulation of reserves to be used in the conversion of theembryos to plantlets. Proteins were predominantly observed in protodermcells, as well as in the meristematic apical region of torpedo and cotyle-donary-stage somatic embryos.

Keywords: Pineapple guava; Acca sellowiana; Feijoa sellowiana; His-tological analysis; Somatic embryogenesis; Protein body; Starch grain.

Abbreviations: 2,4-D 2,4-dichlorophenoxyacetic acid; PAS periodicacid-Schiff reaction; TBO toluidine blue O.

Introduction

In plant cell tissue culture, competent cells are recognizedby their responses to external signals that activate specificdevelopmental pathways (McDaniel 1984). This is demon-strated when an isolated explant that is not intrinsically re-sponsive acquires this competence when it is activated byan inductive signal (Finstad et al. 1993). This suggests thatcell competence may be acquired through a dedifferentia-tion process (Torrey 1977). Regenerative competence is

normally associated with reentry into the mitotic cycle aswell as with alterations in the cell division planes. In somecases, however, in vitro morphogenic competence is notdirectly associated with the level of mitotic activity(Dolezelova et al. 1992).

Somatic embryogenesis is a process through whichbipolar embryos develop from a nonzygotic cell withoutvascular connections with the original tissue. Somatic em-bryogenesis is a multistep regeneration process startingwith the formation of proembryogenic cell masses, fol-lowed by somatic-embryo formation, maturation, desicca-tion, and plant regeneration (von Arnold et al. 2002).Somatic embryos can differentiate either directly or indi-rectly from the explant (Williams and Maheswaran 1986).Indirect somatic embryogenesis arises from undeterminedcells following the formation of a nondifferentiated callus.Distinguishing between direct and indirect somatic em-bryogenesis is, however, a difficult task. In conifers, em-bryogenic calluses consist of proembryogenic masses (vonArnold et al. 2002), which contradicts the criterion of uni-cellular origin. This type of indirect embryogenesis is,however, rarely found in angiosperms (Haccius 1978).

The histological alterations associated with the positionand activity of competent cells during the acquisition of so-matic embryogenic competence has been the subject of sev-eral studies. For example, in hybrid Rosa species, cells onthe periphery of the callus have been observed to undergointernal segmenting divisions and either form somatic em-bryos directly or continue to proliferate forming embryo-genic calluses (Rout et al. 1998). In Feijoa sellowiana, theformation of a dense layer of meristematic cells originating

Protoplasma (2004) 224: 33–40DOI 10.1007/s00709-004-0055-5 PROTOPLASMA

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Morphohistological analysis and histochemistry of Feijoa sellowianasomatic embryogenesis

G. C. Cangahuala-Inocente, N. Steiner, M. Santos, and M. P. Guerra*

Grupo de Pesquisas em Recursos Geneticos Vegetais, Departamento de Fitotecnia, Centro de Ciências Agrárias, Universidade Federal de SantaCatarina, Florianópolis, Santa Catarina

Received February 11, 2004; accepted March 5, 2004; published online October 4, 2004© Springer-Verlag 2004

* Correspondence and reprints: Departamento de Fitotecnia, Centro deCiências Agrárias, Universidade Federal de Santa Catarina, C.P. 476,88034-001 Florianópolis, SC, Brazil.E-mail: [email protected]

in the adaxial face of the cotyledons of zygotic embryos hasbeen described. Two patterns of somatic-embryo differentia-tion were observed: one from single epidermal cells and theother from groups of meristematic cells located near theadaxial surface (Canhoto and Cruz 1996).

The plant growth regulators used for embryogenic inductionproduce alterations in cell polarity and promote subsequentasymmetric divisions (Ammirato 1983). Carya illinoinensiscultures induced by naphthaleneacetic acid have been reportedto show embryogenic regions composed of homogeneous, iso-diametric, meristematic cells, and the somatic embryos derivedfrom these cultures generally had a normal morphology. Incontrast, somatic embryos induced in culture media containing2,4-dichlorophenoxyacetic acid (2,4-D) showed abnormalities(Rodriguez and Wetzstein 1998).

The aim of the present work is to evaluate the morpho-histology and the histochemical aspects associated with theinduction and development of somatic embryos from com-petent explants of Feijoa sellowiana cultured in inductiveconditions.

Material and methods

Plant material

Ripe fruits of Feijoa sellowiana (O. Berg) O. Berg genotype 101 were ob-tained from the germplasm collection of the São Joaquim ExperimentalStation (EPAGRI), Santa Catarina, southern Brazil, and seeds were surfacesterilized according to Guerra et al. (2001). The zygotic embryos were ex-cised in an aseptic chamber and inoculated into test tubes (25 � 150 mm)containing 15 ml of induction medium, consisting of basal medium LPm(von Arnold and Eriksson 1981) supplemented with Morel vitamins (Moreland Wetmore 1951), glutamine (8 mM), 2,4-D (20 �M), sucrose (3%), andagar-agar (0.7%). The pH was adjusted to 5.8 prior to autoclaving. The cul-tures were maintained in the dark at 25 �C during the induction phase.

Microscopic preparation

Zygotic embryos incubated in the induction medium were removedevery 3 days over the 90-day culture period and fixed for 24 h in 0.2 Mphosphate buffer (pH 7.3) containing 2.5% paraformaldehyde. After fix-ation, the samples were dehydrated in a graded ethanol series and em-bedded in historesin (Leica), as described by Arnold et al. (1975).Sections, 5 �m thick, were cut with a rotary microtome (Slee Technik)and fixed onto slides by heating.

Samples were dehydrated with periodic acid and stained by the periodicacid-Schiff reaction (PAS) to reveal starch grain location. Storage proteins

34 G. C. Cangahuala-Inocente et al.: Somatic embryos of Feijoa sellowiana

Fig. 1a–d. Histology of F. sellowiana embryogenic cultures induced by 2,4-D (20 �M). a Longitudinal section of zygotic embryo after 15 days inculture showing large cells in the cotyledonary tissues and small, compact cells in the root tissues. b Cell segregation resulting from proliferativeburst of epidermal cells in zygotic-embryo cotyledon after 18 days in culture. c Induction of meristematic cluster originating from parenchyma cellsof cotyledon after 21 days in culture. d Induction of globular somatic embryos after 60 days in culture. a–c Stained with TBO, d stained with PAS. coCotyledon, ra root, me apical meristem, seg cell segregation, me-no meristematic nodule, se somatic embryo, se-glo globular-stage somatic embryo,st starch. Bars: a, 0.300 mm; b–d, 100 �m

were stained with Coomassie brilliant blue R250 (Sigma) (Gahan 1984),and acid polysaccharides and phenols were stained with 0.5% toluidineblue O (TBO) (O’Brien et al. 1965). Photographs were taken with a stan-dard Olympus BX 40 microscope.

Results

After 15 days on somatic embryogenesis induction medium,zygotic embryos showed expanded, green cotyledons. In thelongitudinal section of the cotyledon, stained with TBO,large cells with parietal nuclei and cytoplasm and just onelarge vacuole could be observed. In contrast, the root re-gions revealed small cells with a high nucleoplasmic ratio,dense cytoplasm, and a small or absent vacuole (Fig.1a).

After 18 days in culture, a proliferative burst in theepidermis and the beginning of cellular segregation couldbe seen (Fig.1b). Cells originating from this processwere small and isodiametric with a parietal nucleus andlarge vacuole and contained phenolic compounds andstarch grains. After 21 days in culture, meristematic cen-ters showing two distinct regions were observed (Fig.1c).One region was centrally located with intense mitotic ac-tivity and protein synthesis, as indicated by Coomassiebrilliant blue R250 staining (data not shown). The secondregion was characterized by the accumulation of phenoliccompounds, as revealed by the green metachromatic re-action (Fig.1c).

G. C. Cangahuala-Inocente et al.: Somatic embryos of Feijoa sellowiana 35

Fig. 2 a–d. Indirect somatic embryogenesisin F. sellowiana. a Somatic embryos arisingfrom a layer of embryogenic cells. b Induc-tion of proembryos. c Group of suspensorcells. b and c Note the presence of polyphe-nols (po). d Fusion of somatic embryos. Allsections were stained with TBO. se Somaticembryo, proder protoderm, seg cell segrega-tion, su-ce suspensor cells, fu fused somaticembryos. Bars: a, 150 �m; b–d, 50 �m

The first visualization of somatic embryogenesis waspossible after 39 days in culture. After 60 days in culture,histological analysis revealed the development of somaticproembryos arising from peripheral cells of the meriste-matic centers (Fig.1d). Protein bodies were observed inthe cells of somatic proembryos (data not shown).

An embryogenic cell layer surrounding the meristematiccenters (Fig. 2a) was competent for somatic-proembryo de-velopment. The first divisions of this cell layer were pericli-nal, but subsequent divisions occurred in several planes. Theproembryos developed from clumps of cells (Fig. 2b, c).Staining with TBO revealed that the cells of this peripherallayer were small and isodiametric, and their vacuoles werefilled with polyphenol compounds (Fig. 2b, c). Once themeristematic centers acquired embryonic features, fragmen-tation of these cellular masses was frequently observed. As a

consequence of this fragmentation, groups of embryoniccells were isolated from the surrounding tissue (Fig. 3a).

The cells of somatic embryos in different developmentalstages showed similar histochemical reactions. However, themorphological features were distinct. The cells of globularsomatic embryos contained few starch grains and were sur-rounded by a layer of protoderm cells. These cells weresmall with high nucleoplasmic ratios and dense cytoplasm(Fig. 3b). In the early torpedo stage, the metachromatic reac-tion of TBO was observed specifically in the basal cells(Fig. 3c), similar to the observed pattern in the cells of theperipheral layer surrounding the meristematic centers (seeFig.1c). Starch grains were also present in these cells butwere absent from the apical region of somatic embryos(Fig. 3d). A positive Schiff reaction also revealed starchgrains in the intracellular domain of basal cells of torpedo


Fig. 3a–f. Histological sections of F. sell-owiana somatic embryos. a and b Em-bryogenic cells forming globular somaticembryos. Note the presence of polyphenols(po) and starch granules (st) in the mothercells. b Globular somatic embryos showinga well-developed protoderm. c and d Early-torpedo-stage somatic embryos. e and fTorpedo stage somatic embryos showing pro-cambial region. a and c Stained with TBO. b,d, and e Stained with PAS. f Stained withCoomassie brilliant blue. proder Protoderm,procam procambium, pro protein body, segcell segregation. Bars: a–e, 50 �m; f, 100 �m

stage embryos (Fig. 3e). Coomassie brilliant blue staining re-vealed protein bodies in all cells at this stage (Fig. 3f). Acidpolysaccharides could be seen in pre-cotyledonary-stage so-matic embryos stained with TBO, mainly as constituentsof the cellular wall (Fig. 4a, b). Cotyledonary-stage somaticembryos contained protein bodies in the protoderm cells(Fig. 4d), as well as starch grains in the basal cells (Fig. 4e).

Somatic embryos in the early torpedo (Fig. 3c), torpedo(Fig. 3f), pre-cotyledonary (Fig. 4a, b), and cotyledonarystages (Fig. 4c) exhibited differentiated regions contain-

ing protoderm and procambial cells. These somatic em-bryos also revealed conspicuous apical and root meristemregions. Initially, somatic embryo development was syn-chronous (Fig.1d), but continued in an asynchronousmanner (Fig. 2a). Vascular connections were detected be-tween the embryos and the peripheral cells (Fig. 2a). Ab-normalities were often found in the developing somaticembryos, such as an altered number of cotyledons and,most commonly, the presence of fused somatic embryos(Fig. 2d).


Fig. 4a–f. Histological sections of F. sello-wiana somatic embryos. a and b Pre-cotyle-donary somatic embryos showing protodermand procambial strands. c–e Cotyledonarysomatic embryos. f Cell agglomerates withstarch grains. a–c Stained with TBO. d Stainedwith Coomassie brilliant blue. e and f Stainedwith PAS. proder Protoderm, procam procam-bium, co cotyledon, po polyphenols, pro pro-tein bodies, st starch granule. Bars: a–e,100 �m; f, 50 �m

Discussion

Development of somatic embryogenesis

In the present work, somatic embryos differentiated denovo from the segregation of cotyledon cells of zygoticembryos. This process occurred in two steps: first, cellularsegregation originating in meristematic centers; second,formation of a peripheral cell layer surrounding the meri-stematic centers, with every cell of this layer showingcompetence for somatic embryogenesis.

The cells resulting from the segregation were isodiamet-ric, with a parietal nucleus and a large vacuole, and con-tained phenolic compounds and starch grains. It has beenpreviously reported that single cells can produce few-celledproembryos, referred to as embryogenic units in Zea mays(Fransz and Schel 1991) or proembryonic cell masses inPennsisetum glaucum (Taylor and Vasil 1996) and Quercussuber L. (Puigderrajols et al. 2001).

In the present work we observed the presence of thick cellwalls surrounding the proembryo cells. Small globular clus-ters without visible polarity were associated with earlierproembryos, whereas globular clusters in which polarity wasalready established were associated with later developmentalstages. Similar morphogenetic features have been describedfor Guinea grass by Karlsson and Vasil (1986) and for corkoak by Puigderrajols et al. (2001).

The development of zygotic embryos is well under-stood since they originate from the fusion of two haploidcells. The origin of somatic embryos has been associatedwith two pathways: unicellular or multicellular. Unicellu-lar somatic embryogenesis results from the developmentof single cells, whereas multicellular somatic embryogen-esis results from the association of embryogenic cells orevolves from embryogenic cell clusters (Michaux-Ferrièreand Schwendiman 1993).

Our results suggest that the somatic embryos in thisstudy had both unicellular and multicellular origins, as hasbeen described for other dicotyledonous species (Colbyet al. 1991). For Panicum maximum, it has been demon-strated that somatic embryos arise from single cells andclosely resemble the developmental morphology of zygoticembryos of grasses (Botti and Vasil 1984, Lu and Vasil1985). Canhoto and Cruz (1996) have shown that somaticembryos of F. sellowiana can arise directly from multi-cellular cell clumps on the epidermal adaxial surface of zygotic embryo cotyledons. Somatic embryos of F.sellowiana have also been observed to arise directly fromthe cotyledonary tissues of zygotic embryos after sixteendays in culture (Guerra et al. 2001).

Storage products

Reserve compounds play an important role in in vitro mor-phogenesis. For example, high levels of polysaccharides atthe beginning of the in vitro developmental process havebeen reported (Branca et al. 1994), and the consumption ofthese compounds has been correlated with the onset oforganogenesis and somatic embryogenesis (Mangat et al.1990, Martin et al. 2000).

In Carya illinoinensis, the formation of embryogenic pro-tuberances is preceded by the accumulation of starch gran-ules in the subepidermal cell layers of the explant. Starch israpidly consumed during the formation of embryogenic re-gions and is absent from globular and heart-shaped embryos(Rodriguez and Wetzstein 1998). Our results are in agree-ment with these findings since the meristematic centers con-tained abundant starch grains which were heavily depletedin the proembryonic cell clumps. Starch is considered to bethe primary source of energy for cellular proliferation andgrowth. The consumption of these starch grains, therefore,should provide energy for the development of the somaticembryos, suggesting an active regulation of starch accumu-lation as has been proposed by Martin et al. (2000). Canhotoand Cruz (1996) could not detect starch grains in meriste-matic layers of F. sellowiana; although, they were present inproembryos. This suggests that starch is rapidly metabolizedin embryogenic tissues, providing energy for the intensemetabolic and mitotic activity (Stamp 1987).

Our histochemical evaluations also revealed that embryo-genic cells resulting from cell segregation contain proteinbodies, which were also observed in the meristematic cen-ters. Most seed storage proteins are secretory proteins syn-thesized from a peptide that is cleaved as the protein istransported into the lumen of the endoplasmic reticulum(Shewry et al. 1995). Storage proteins found in vacuoles arespherical protein bodies that are degraded during germina-tion to provide carbon and nitrogen for the growing seedling(Shotwell and Larkins 1989). It has been suggested that thepresence of proteins in the embryogenic cells is associatedwith the formation of proembryonic cell groups. Meriste-matic centers formed of isodiametric cells with prominentnucleoli and high mitotic activity have been observed inEucalyptus urophylla. A well-defined surrounding cellularregion could be stained with naphthol blue-black, revealingsites of protein synthesis (Arruda et al. 2000).

A remarkable feature of the meristematic centers that pro-duced the somatic embryos was the presence of polyphenoliccompounds. The cultures also showed an enhanced produc-tion of brown exudates of polyphenol origin. These com-pounds appeared to inhibit hyperhydricity, thereby serving as


precursors of lignin synthesis as has been observed in Euca-lyptus nitens (Bandyopadhyay and Hamill 2000). Cells thatstain green with the metachromatic stain TBO are reported tocontain polyphenols or lignin (Feder and O’Brien 1968).

Histochemistry of somatic embryos

As stated previously, we could observe four stages ofsomatic-embryo development. The beginning of histogene-sis could be seen at the globular stage with the formationof protoderm surrounding the globular embryo (Yeung1995). This feature could be clearly observed and was as-sociated with the presence of few starch grains but abun-dant protein bodies.

The methods used to identify the different types of poly-saccharides revealed different regions of acid and neutralcompound accumulation in F. sellowiana somatic embryos.Acid polysaccharides, indicated by the metachromatic stainTBO, were observed mainly as constituents of the cellularwall, whereas neutral polysaccharides, indicated by theSchiff reaction, were mainly observed as reserve substancesin the intracellular domain. The Schiff reaction identifiesneutral polysaccharides due to its requirement for the pres-ence of 1,2-glycol groups that are oxidized by periodic acid(Trick and Pueschel 1990).

Histochemical evaluations of the torpedo and cotyle-donary-stage somatic embryos revealed that starch grainsoccurred only in the root region, while protein bodies wereobserved in the protoderm as well as in the apical meri-stem. Borisjuk et al. (1995) have reported the presence ofsmall and large starch grains in the protoderm and cotyle-donary cells, respectively, of Vicia faba zygotic embryos.

The morphological abnormalities of the somatic embryosobserved in this work could be due to the 2,4-D supplementin the induction medium. The high incidence of abnormalembryos in this species has also been attributed to their mul-ticellular origin (Canhoto and Cruz 1996). Somatic embryosof Carya illinoinensis induced with 2,4-D have also beenobserved to show a high incidence of abnormalities includ-ing fasciated, fan-shaped, and tubular embryos (Rodriguezand Wetztein 1998).

In conclusion, the results of the present work show that so-matic embryos of F. sellowiana can be induced from embryo-genic cells arising in meristematic centers or from clusters ofisolated cells. The presence of few starch grains and abundantprotein bodies was a distinctive feature of globular somaticembryos, while cells in the torpedo and cotyledonary stagescontained abundant starch grains. This could be associatedwith the strategy of storing reserve compounds to be used inthe further conversion of the embryos to plantlets.

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morphohistological analysis and histochemistry of feijoa sellowiana

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