Biologia 66/2: 229—237, 2011Section BotanyDOI: 10.2478/s11756-011-0007-4

Comparative study of gametophyte developmentin the some species of the genus Onobrychis:Systematic significance of gametophyte futures

Abdilkarim Chehregani*, Fariba Mohsenzadeh & Nayereh Tanaomi

Laboratory of Plant Cell Biology, Department of Biology, Bu-Ali Sina University, Hamedan, Iran;e-mail: chehregani@basu.ac.ir

Abstract: Male and female gametophytes have special characters that show a great variety in different taxa. In this study,gametophytes of four species belonging to three sections of the genus Onobrychis Mill. were studied with light microscopy.Results showed that the ovular primordium is tetra-zonate and gives rise to an anatropous ovule. The archesporium mayconsist of one or more archeosporial cells, but only one of them undergoes meiosis, forming a linear or T-shaped tetrad.Normally, only a single megaspore is functional which is located in the chalazal position while the others degenerate verysoon. The young ovule is hemi-anatropous but the mature is anatropous, crassinucellar and bitegmic; integuments form azig-zag micropyle. A 7-celled embryo-sac is formed corresponding to the Polygonum type. Based on our results, the ovularvariable characters are the form and condition of ovary, presence or absence of ovary peduncle, the number and condition ofovule in ovary, length and width of ovule, length and width of embryo sac, number of layers in outer integument, conditionof megaspore, alignment pattern of the integuments, asymmetrical initiation of the outer integument, shape of tetrad withthe presence of one functional megaspore and so on. The separator characters in male gametophyte are including tri-cellularpollen grains and the number of tapetum nuclei. According to our study the female gametophyte characters are morevariable than male gametophyte. The present study provides the first report on embryological description in the genusOnobrychis and also in section Heliobrychis.

Key words: Onobrychis schahuensis; O. andalanica; O. melanotricha; O. altissima; Fabaceae; microgametophyte; megaga-metophyte; ovule; pollen

Introduction

The genus Onobrychis belongs to tribe Hedysareae,from Fabaceae family, with nearly 130 species is mainlydistributed in the northern temperate regions but cen-ters of it’s diversity is in the eastern Mediterranean areaandWest Asia; a few taxa are cultivated as fodder or or-namentals (Lock & Simpson 1991; Yakovlev et al. 1996;Mabberley 1997). In Flora Iranica, Rechinger (1984)treated 77 species under 9 sections, viz. Dendrobrychis(7), Lophobrychis (5), Onobrychis (14) Laxiflorae (3),Anthyllium (7), Afghanicae (3), Insignes (3), Helio-brychis (21) and Hymenobrychis (12 species), with twospecies remaining unassigned. Morphological charactersof the ovules and megasporogenesis stages can be usedin systematic studies for defining the circumscription oftaxa (Rembert Junior 1966). Male and female gameto-phytes have special characters that show a great vari-ety in different taxa (Johri et al. 1992). The charactersof these organs can be used in separating and definingtaxa position. Although pollen morphology was used asthe most important microscopic character in the tax-

onomy of Onobrychoidei (Pinar et al. 2009) but thereare also some reports about taxonomically importantof ovule characters in the Fabaceae family such as vari-ability in the megaspore tetrads, ovule type, numberof archesporial cells, number of parietal layers and thealignment pattern of the integuments (Rembert Junior1966, 1969a, b, 1971; Davis 1966; Prakash 1987; Yeung& Cavey 1990; Johri et al. 1992; Dute & Peterson 1992;Chamberlin et al. 1994; Johansson & Walles 1993; Ri-ahi et al. 2003; Chehregani & Tanaomi 2010), but Ono-brychis is still embryologically unknown.The aim of this work was to compare gametophyte

development in four species belongs to three sections ofthe genus Onobrychis and evaluate taxonomical signif-icance of gametophyte characters that can be useful inseparating and defining taxa position.

Material and methods

The plants used in this study were collected from the nat-urally growing area. Voucher specimens are deposited at

* Corresponding author

c©2011 Institute of Botany, Slovak Academy of Sciences

230 A. Chehregani et al.

Table 1. The information of the studied species that were subjected for embryological investigation in this study.

No. Species Voucher number Altitude Date Collector Location

1 O. altissima Grossh. (BASUH) (11001) 1930 m 3.5.2008 Ranjbar East Azerbaijan, Road ofVarzaghan to Tabriz, Km 10,Mirza-Ali Village

2 O. andalanica Bornm. (BASUH) (11000) 1200 m 2.4.2007 Ranjbar Kordestan, Road of Kam-yaran to Mochesh, 17 Km toMochesh

3 O. melanotricha var. villosa Bornm. (BASUH) (10999) 1600 m 2.4.2007 Ranjbar Kordestan, Ghorveh toSanandaj, near to Dehkolan,Hasan-Abad Village

4 Onobrychis schahuensis Bornm. (BASUH) (10998) 1250 m 22.3.2007 Chehregani Kermanshah, Road of Javan-roud to Tazeh Abad, Km 15

the herbarium of the Bu-Ali Sina University (HBAS). Thevoucher specimens are labeled in Table 1.

The flowers and young buds were removed in differ-ent developmental stages, fixed in FAA70 (formalin, glacialacetic acid and 70% ethanol, 5:5:90 v/v), stored in 70%ethanol, embedded in paraffin and sectioned at 7–10 µmwith a Micro DC 4055 microtome. Staining was carried outwith PAS (Periodic Acid Schiff) according to protocol sug-gested by Yeung (1984) and contrasted with Meyer’s Hema-toxylin (Chehregani et al. 2009). Several sections were stud-ied for each ovule and pollen developmental stages under aZeiss Axiostar Plus light microscope. For each stage, at least20 flowers were studied and photomicrographs made fromthe best ones. The developmental stages were compared inthe studied taxa for finding significant characters that areuseful in taxonomy.

Results

Female gametophyteResults showed that in O. schahuensis and in all stud-ied species ovule initiation is basipetal and originates asa small protuberance (Fig. 1). The initial archeosporialcell is distinguished from the other sub-dermal cells,because it presents a larger volume, dense cytoplasmand distinct nucleolus (Fig. 2). One of the archespo-rial cells act as megasporocyte or megaspore mothercell (Fig. 3). The megaspore mother cell (MMC) di-vides meiotically and undergoes two successive divi-sions. Meiosis I forms dyad cells (Fig. 4), and meio-sis II forms tetrad cells (Fig. 5). The three micropylarmegaspores degenerate, and the chalazal one is func-tional megaspore and develops into the megagameto-phyte (Fig. 6). Three successive mitotic karyokinesisgive rise to an eight-nucleate embryo-sac (Fig. 7). Acentral vacuole is formed and four nuclei are positionedin the micropylar end of the embryo sac, and the otherfour nuclei in the chalazal end. After the eight-nucleatestage, the coenocytic megagametophyte becomes partlycellular. This process is simultaneous at the micropylarand chalazal ends. The embryo-sac in all species con-sists of seven cells: the egg cell and two synergids thatformed egg apparatus, the central cell that is the largestcell and three antipodal cells (Figs 8, 9). Such an em-bryo sac represented the Polygonum type. In the allspecies central cell that is the largest cell of the embryo

sac, contains two polar nuclei (Figs. 8, 9) and antipodalcells are the smallest cells of the embryo sac (Figs 8, 9).In the all species when the embryo sac starts

its development, the ovule is completely inverted, sothe nucellus and integuments lie alongside the funicule(Fig. 10) and the outer integument develops first. Si-multaneously with the development of the embryo-sac, a structure consisting of nucellar tissue resistantto the absorbing activity of the embryo sac is ob-served. An endothelium is originated from the innerlayer of the inner integument. The cells of this layerbecome radially stretched and they contain promi-nent nuclei and dense cytoplasm (Fig. 11). The ma-ture ovule is anatropous, crassinucellar, bitegmic witha zig-zag micropyle and the carpel is already closed inall species, when the first ovule primordium appears(Fig. 12).

Male gametophyteIn the all species the undifferentiated anther is ovoid-shaped and tetrasporangiated (Fig. 13). The antherwall consists of epidermis, endothecium, one middlelayer and tapetum that is secretory type (Figs 13–18).The microsporangial wall follows the dicotyledonoustype of ontogeny. The outer secondary parietal layergives rise to two layers, the outermost forming, the en-dothecium, and the innermost, the middle layer. Theinner layer directly functions as tapetum. The earlymicrospore mother cells are nucleated and poorly vac-uolated (Fig. 13). Then late microspore mother cellsbecome separated and each of them is surroundedby a callose coat (Fig. 14). In this time tapetum iswell-differentiated. When microspore mother cells en-tered the phase of meiotic division, the four haploidmicrospores become enclosed in a thick callose wall(Fig. 15). At the early tetrad stage, the microsporescontain a big nucleus at the center (Fig. 15). The mi-crospore tetrads are still within the callose walls duringthe late tetrad stage (Fig. 16). Once released from thetetrad, the microspores enlarged in size (Fig. 17). Acentral vacuole appears in them, pushing the nucleustowards the periphery (Fig.17). At this time, degen-eration of tapetum is visible well (Fig. 18). Maturepollen grains were ovate (Fig. 19). As a result, the mi-totic division of the microspore is unequal. Therefore,

Gametophyte development in some species of the genus Onobrychis 231

Figs 1–10. Gametophyte development in Onobrychis schahuensis. 1 – Longitudinal section of a young carpel with the first (upper)ovule primordium; 2 – The ovule originates as a small protuberance and the initial archesporial cell (↑) is distinguished from theother sub-dermal cells. Outer (oi) and inner integument (ii) are initiated; 3 – Young ovule with prominent megaspore mother cell(Mg). The two integuments (↑↑) are initiated from periclinal and oblique divisions of dermal cells; 4 – Young ovule with a megasporesmother cell (↑, Mg), the outer integument (oi) grows faster than inner one (ii); 5 – Linear arrangement of megaspore tetrads (Te); 6 –One-nucleated embryo sac (↑). The inner integument differentiates simultaneously as a ring around the nucellus; 7 – Embryo sac duringcellularization (arrows) with a big vacuole (v); 8 – Egg apparatus (eg) and secondary nucleus (↑) resulted by the fusion of polar nuclei(pn) in the micropylar end; 9 – Mature embryo sac with differentiated egg apparatus and secondary nucleus (↑). Cellularization of thecells was completed but two polar nuclei were not combined yet; 10 – Mature embryo sac with fused polar nuclei as the secondarynucleus (↑). Synergids and oospher (egg apparatus) are visible in the micropylar end of embryo sac (eg).

a small generative cell and a large vegetative cell ap-pear so pollen grains have two-celled at the time ofshedding (Fig. 19). In this time tapetum degeneratecompletely and we can see endothecium and epider-mis.

Specific gametophyte features in the speciesAlthough the overall pattern of gametophyte develop-ment is similar in the all studied species and is accor-dance with above mentioned pattern. But there are alsosome differences between the species. Species-specificfutures of female and male gametophyte developmentwill present as following.

Female gametophyte in Onobrychis schahuensisBornm.The ovary is curved, without peduncle and hair. Thecarpel is already closed when the first ovule primordiumappears. The inner integument is two cells thick anddifferentiates simultaneously as a ring around the nu-cellus where the outer integument is 4–5 cells thickness(Figs 4, 6) and on the opposite side of the funicule,grows asymmetrically faster. The number of cell lay-ers in the nucellar tissue is about 18–22 layers that it’sthe most between our species. In O. schahuensis thedyad cells have unequal size and the tetrad cells arelinear type (Fig. 5). The embryo sac length is about

232 A. Chehregani et al.

Figs 11–19. Gametophyte development in Onobrychis schahuensis. 11 – In this view three antipodal cells (↑) are evidence in thechalazal end of embryo sac, opposite to micropyle (m); 12 – Mature ovule with showing zig-zag micropyle (↑). Two anatropous,crassinucellar and bitegmic ovules with a zig-zag micropyle were seen in the young ovary but upper ovule is degenerative ones andmature legume contains only one seed; 13 – Young anther is ovoid-shaped and tetrasporangiated. The anther wall consists of epidermis(E), endothecium (EN), one middle layer (ML) and tapetum (T). Pollen mother cells (PMC) are large nucleated, poorly vacuolatedand located at the center of anther; 14 – Late microspore mother cells (MC) become separated and each of them is surrounded bya callose coat; 15 – Early tetrad stage (↑) surrounded by callosic wall (c). The microspores contain a big nucleus at the center; 16 –Late tetrad stage, the microspore tetrads (↑) are still within the callose (c); 17 – Released microspores (↑), with a central growingvacuole that pushing the nucleus toward peripherally; 18 – Mature pollen grain in polar and equatorial view at the shedding time; 19– Mature pollen grains (↑) with generative and vegetative nuclei.

430 µm. The fusion position of polar nuclei, after for-mation in chalazal pole and emigration to micropylarpole, is near the egg apparatus (Fig. 8). Large amy-loplasts, darkly stained, are also seen in the embryo-sac but the amount of them is less than other species.The ovule length is about 630 µm and the width of it,is about 320 µm. Young legumes have two ovules inearly developmental stages that seem be synchronous,but upper ovule degenerates before fertilization. Thus,mature legumes contain only one seed. Degenerationof upper ovule takes place slowly but its evidences arevisible (Fig. 12).Male gametophyte in O. schahuensis Bornm.InO. schahuensis the number of pollen grains is about25–30 in each sporangium, and the polar length of themis about 29 µm. The thickness of endothecium is about18 µm. The tapetal cells are uni-nucleate but some ofthem are bi-nucleate. Cytokinesis is simultaneous andthe arrangement of microspores is tetrahedral (Fig. 15).Female gametophyte in O. andalanica Bornm.In this species the young ovary have one ovule andthus the mature legumes contain only one seed too.

The ovary is curved, with a long peduncle and hairs.When the first ovule primordium appears, the carpel isalready closed. The inner integument is two cells thick(Fig. 20) where as the outer integument is 3 cells thick(Fig. 20). On the opposite side of the funicule, the outerintegument grows asymmetrically slower (Fig. 21). Inthe nucellar tissue, the number of cells is about 12–14layers. The dyad cells have equal size and the tetradcells are T shaped (Fig. 22). The embryo sac length isabout 570 µm. The fusion position of polar nuclei is im-mediately after formation and it is in chalazal pole. Thenumber of amyloplast that accumulated starch grainsis too much in the cytoplasm of embryo sac. The ovulelength is about 700 µm and the width of it, is about380 µm.Male gametophyte in O. andalanica Bornm.The number of pollen grains is about 51–53 and thepolar length of them is about 25 µm. The thicknessof endothecium is about 19 µm. The tapetal cells areuni-nucleate. Cytokinesis is simultaneous and the ar-rangement of microspores is tetrahedral and tetragonal(Fig. 23).

Gametophyte development in some species of the genus Onobrychis 233

Figs 20–23. Gametophyte in Onobrychis andalanica. 20 – Young ovule with one-nucleated embryo sac (↑) in O. andalanica. Theinner integument is two-celled thicker and outer integument is three-celled thicker; 21 – On the opposite side of the funicule, theouter integument grows asymmetrically slower; 22 – Megaspore tetrad is T-Shaped (↑); 23 – Anther having tetrahedral and tetragonalmicrospore tetrads (↑).

Figs 24–29. Gametophyte in Onobrychis melanotricha (24–26) and Onobrychis altissima. (27–29). 24 – Young ovary contained ayoung ovule at dyad stage (↑); 25 – The fusion position of polar nuclei is in chalazal pole and then emigrated to micropyle (↑); 26 –Pollen anther at the microspore tetrad stage. Tetrad arrangement is both tetrahedral and tetragonal ones (↑); 27 – Young ovule in O.altissima. The megaspore mother cell is in the second layer; 28 – The inner integument is two-celled thicker and outer integument is4–5 cells thick; 29 – The fusion position of polar nuclei (↑), after formation in chalazal pole, is in micropylar end.

Female gametophyte in O. melanotrichavar. villosa Bornm.In this species, young ovaries have only one ovule andthe mature legumes contained only one seed too. Theovary is nearly right, with peduncle and hairs. Whenthe first ovule primordium appears, the carpel is alreadyclosed. The megaspore mother cell is in the fifth layer.The inner integument is two cells thick where the outerintegument is 3 cells thick (Fig. 24). On the oppositeside of the funicul, the outer integument grows asym-metrically slower. In the nucellar tissue, the number of

cells is about 10–12 layers. The dyad cells have equalsize (Fig. 24) and the tetrad cells are T-shaped. The em-bryo sac length is about 400 µm. The fusion positionof polar nuclei is in chalazal pole, immediately afterformation (Fig. 25). Large amyloplasts, darkly stained,are also seen widely in the placental cells. The Ovulelength is about 560 µm and the width of it, is about310 µm.Male gametophyte in O. melanotrichavar. villosa Bornm.The number of pollen grains is about 54–55 in each

234 A. Chehregani et al.

sporangium and the polar length of them is about 32µm. The thickness of endothecium is about 18 µm. Thetapetal cells are uninucleate. Cytokinesis is simultane-ous and the arrangement of microspores is tetrahedraland tetragonal (Fig. 26).Female gametophyte in O. altissima Grossh.The young ovaries have only one ovule and the maturelegumes contain only one seed too. The ovary is nearlyright, with peduncle and hairs. When the first ovuleprimordium appears the carpel is already closed. Themegaspore mother cell is in the second layer (Fig. 27).The inner integument is two cells thick where as theouter integument is 4–5 cells thick (Fig. 28). In the nu-cellar tissue, the number of cells is about 15–16 layers.The dyad cells have equal size (Fig. 28) and the tetradcells are T-shaped. The embryo sac length is about 370µm. The fusion position of polar nuclei, after formationin chalazal pole (Fig. 29) and emigration to micropylepole, is near the egg apparatus. The amyloplasts accu-mulation in cytoplasm of embryo-sac is too much. Theovule length is about 480 µm and the width of it, isabout 280 µm.Male gametophyte in O. altissima Grossh.The number of pollen grains is about 63–67 and thepolar length of them is about 31 µm. The thickness ofendothecium is about 13 µm. The tapetal cells are un-inucleate. Cytokinesis is simultaneous and the arrange-ment of microspores is tetrahedral and tetragonal.

Discussion

Bouman (1971, 1974, 1984) compared cellular organi-zation in the ovule primordium with the shoot apicalmeristem. Our results showed that in the all studiedspecies, ovule initiation is basipetal and originates asa small protuberance. The ovular primordium in allspecies is tetra-zonate. The ovule is anatropous ac-cording to the ontogenetic classification of Bocquet &Bersier (1960). This anatropous curvature, as in otherlegumes, is usually related to unequal growth at funic-ular region (Bocquet, 1959; Bocquet & Bersier 1960;Bor 1978; Bouman & Boesewinkel 1991). Some au-thors classified this type of ovule as campylotropous(Reeves 1930; Pal 1960; Rembert Junior 1967; Ojeaga &Sanyaolu, 1970; Oomman, 1971; Deshpande & Bhasin,1976) or anacampytropous (Prakash & Chan, 1976).One of the important characters of the ovules is

the number of cell layers in each integument. The in-ner integument in the all studied species as in the mostFabaceae, consists of two cell layers (Cooper 1933; Roy1933; Samal 1936; Paul 1937; Smith 1956; Dnyansagar1957; Hindmarsh 1964; Deshpande & Bhasin 1976;Rembert Junior 1977; Ashrafunnisa & Pullaiah 1994,1999). However, in Glycine javanica L., Tephrosia, Cli-toria ternata L., Pongamia glabra Vent. (AnantaswamyRau 1951), Teramnus labialis (L.f.) Spreng. (Anan-taswamy Rau 1953), Psophocarpus tetragonolobus (L.)DC. (Lim & Prakash 1994) and also in the species ofCassia (Pantulu 1945) it is formed by more than twolayers. An outer integument with several cell layers is

common in Fabaceae. As in the ovules of O. andalan-ica and O. melanotricha it was consisted of 3 layers butbased on our results, in O. altissima and O. schahuensisit was 4–5 layered. In a few Mimosoideae (Dnyansagar1954) and Glycine max L. (Prakash & Chan 1976) theouter integument has only two layers. The outer integu-ment on the opposite side of the funicular region, in O.andalanica and O. melanotricha grows asymmetricallyslower but in O. schahuensis and O. altissima, it growsasymmetrically faster.The nucellus of ovules in Fabaceae is crassinucel-

late (Prakash 1987; Faigón Soverna et al. 2003; Moco& Mariath 2003; Galati et al. 2006), which were alsofound in our species. This type has been considered tobe the plesiomorphic condition in angiosperms (Sporne1969). The number of cell layers in this tissue is alsovariable in the mature ovules. Our results showed thepresence of about 12–14 layers in O. andalanica, 10–12layers in O. melanotricha, 15–16 layers in O. altissimaand 18–22 layers in O. schahuensis. The definitions ofnucellus types, however, do not take into account someaspects & Rutishauser (1982) considered to be impor-tant in the evolution of the archesporium, such as theorigin of its cells and the moment when the vegetativepotential is converted to a reproductive one, as previ-ously emphasized by Warming (1878).Our results showed that the megagametocyt posi-

tion in O. schahuensis, O. andalanica and O. altissimais in the fourth layer but in O. melanotricha it is in thefifth layer (Table 2). The dyad cells have equal size in O.andalanica, O. melanotricha and O. altissima but areunequal in O. schahuensis. Studies of megasporogenesisin the Fabaceae have shown great variability in tetradshape. Our results indicate the presence of T-shapedtetrad in O. andalanica, O. melanotricha and O. al-tissima and the linear shaped tetrad in O. schahuensis(Table 2). T-shaped tetrads were also reported in themost of legumes (Chehregani & Majd 1992; FaingónSoverna et al. 2003; Riahi et al. 2003).Our results indicate that functional megaspore

in O. schahuensis is chalazal ones (Figs 8, 9). Hind-marsh (1964) recorded vacuolation in all four megas-pores of the tetrad in Trifolium pratense, but only thechalazal one became larger, while the others degener-ated. In Cassia abbreviata Oliv. the chalazal megas-pore is functional but the epichalazal one persists fora while and is the last one to degenerate (RembertJunior 1969a, 1971).Variations in the position of thefunctional megaspore have been reported in Trifoliumrepens (Martin 1914) and Vicia faba L. (Mitchell 1975)with an epichalazal position, and in Milletia ovalifoliaKurz (Pal 1960), Trifolium hybridum (Kazimierski &Kasimierski 1979) and in some Australian species of thetribe Mirbelieae (Cameron & Prakash, 1994), where itmay be in a chalazal, micropylar or epichalazal position,that functional megaspore in our all studied species ischalazal one.In all studied species, the embryo-sac consists of

seven cells: the egg cell, two synergids, the central celland three antipodal cells (Figs 13–16). Such an embryo-

Gametophyte development in some species of the genus Onobrychis 235

Table 2. The comparison of some separator gametophyte characters in the four species of Onobrychis.

Num Characters O. altissima O. melanotricha O. andalanica O. schahuensis

1 Ovule number 1 1 1 22 Ovule length 480µm 560 µm 700 µm 630 µm3 Ovule width 280 µm 310 µm 380 µm 320 µm4 Embryo sac length 370 µm 400 µm 570 µm 430 µm5 Ovary hair + + + –6 Number of nucellus

layer in the wide ofembryo-sac

15–16 10–12 12–14 18–22

7 Position ofmegagametocyte

Fourth layer Fifth layer Fourth layer Fourth layer

8 Form of megasporetetrads

T-shaped T-shaped T-shaped linear shaped

9 Ovary peduncle + + + –10 Fusion time of polar

nucleiAfter emigration tomicropylar end

Immediately afterformation

Immediately afterformation

After emigration tomicropylar end

11 Fusion position ofpolar nuclei

Beside the eggapparatus

Chalazal pole Chalazal pole Near the eggapparatus

12 Amyloplastaccumulation inembryo sac

Too much Too much Much Few

13 Growth pattern ofinteguments

grow faster onopposite side of funicul

grow slower onopposite side of funicul

grow slower onopposite side of funicul

grows faster onopposite side of funicul

14 Number of layers inouter integuments

4–5 3 3 4–5

15 Number of nuclei intapetal cells

1 1 1 1–2

16 Number of pollengrains in each anther

63–67 54–55 51–53 25–30

17 Polar length of maturepollen grains

31 µm 32 µm 25 µm 29 µm

18 Endothecium thickness 13 µm 18 µm 19 µm 18 µm

sac represents the Polygonum type that is common inthis family (Kazimierski & Kazimierski 1979; Kennel& Horner 1985; Chehregani & Majd 1992; Cameron &Prakash 1994; Rriahi et al. 2003; Galati et al. 2006;Bakar Buyukkartal 2009). The embryo sac length inO. schahuensis, O. andalanica, O. melanotricha andO. altissima is 430 µmm, 570 µmm, 400 µmm and 370µmm, respectively.In O. schahuensis and O. altissima, the polar nu-

clei, after formation in chalazal pole, emigrate to mi-cropylar pole and the fusion position of polar nuclei isnear the egg apparatus. But in O. melanotricha andO. andalanica, polar nuclei were formed in the chalazalpole and then emigrated to micropylar pole and fusionwas done at near the egg apparatus.In O. andalanica, O. melanotricha and O. al-

tissima the ovary has long peduncle and hairs but inO. schahuensis it is without peduncle and hair. Ourresults showed that young ovary contained one ovulein O. andalanica, O. melanotricha and O. altissima. InO. schahuensis young ovary contained two ovules butupper ovule is degenerative one, thus mature legumecontained only one seed in the all samples. Seed abor-tion was also reported in the some other members ofthis family that take placed after fertilization (Arathiet al. 1999). Seed abortion seems therefore to be a re-sult of competition between the two seeds for mater-

nal resources. The evolutionary significance of single-seeded pods in Pongamia pinnata is discussed with re-spect to possible dispersal advantage enjoyed by suchpods (Arathi et al. 1999). Algan & Bakar (1990) re-ported that seed abortion in tetraploid Trifolium pre-tense might be due to several factors. There may besome problems in microspore and megaspore formationdue to difficulties in male and female gametophyte orin the fertilization and post-fertilization stages.The ovular length in O. schahuensis, O. andalan-

ica, O. melanotricha and O. altissima is 630 µm, 700µm, 560 µm and 480 µm, respectively, and the widthof them is 320 µm, 380 µm, 310 µm and 280 µm.The number of pollen grains in each anther is vari-

able: inO. schahuensis, O. andalanica, O. melanotrichaand O. altissima is 25–30, 51–53, 55–54 and 63–67,respectively. The polar length of pollen grains in O.schahuensis, O. andalanica, O. melanotricha and O.altissima is 29 µm, 25 µm, 32 µm and 31 µm, respec-tively. The thickness of endothecium in O. schahuensis,O. andalanica, O. melanotricha and O. altissima is 18µm, 19 µm, 18 µm and 13 µm. The tapetal cells areuni-nucleate in O. andalanica, O. melanotricha and O.altissima but in O. schahuensis they are both bi anduni-nucleate. The comparisons of the most importantcharacters are presented in Table 1. Although manystudies showed that anatomical characters have taxo-

236 A. Chehregani et al.

nomic importance (Abdel-Khalik et al. 2008 and refer-ences therein) but the information presented in table 1is the first report of taxonomical importance of ovuleand anther structure. Prior researches focused only onthe importance of pollen morphology in Fabaceae fam-ily (Pinar et al. 2009). Based on our bibliographicallystudies this is also the first report about comparativestudy on ontogeny of the ovule and anther, the microand megasporogenesis and also the male and female ga-metophyte development in the genus Onobrychis.

Acknowledgements

This research was supported by the grant provided by theResearch Council of Bu-Ali Sina University. The authorswish to thanks Dr. Massoud Ranjbar for providing the ex-perimental specimens and also his valuable comments.

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Received January 5, 2009Accepted August 16, 2010