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Article DOi: 10.5504/bbeq.2011.0031 b&e

Biotechnol. & Biotechnol. eq. 2011, 25(2), 2379-2382 Keywords: lM, pollen ornamentation, seed sculpture, SeM, Tribulus terrestris

IntroductionTribulus l. is a polymorphous genus of the family Zygophyllaceae R. Br. that includes 25 species (27), widely distributed in arid, warm and moderate temperate regions in the world. Squires (23) commented that this species, originating in the Saharan region, spreading into the Mediterranean region, is now nearly a cosmopolitan due to its easy seed transfer.

Tribulus terrestris l. is a cross-pollinated species by insects, along with being self-pollinated (21). the effective realization of the reproductive capacity of this species is also due to its characteristic as an entomophilous plant which produces a large quantity of pollen grains. the character of the fruit of T. terrestris, with clearly expressed spiny segments, determines the reason it reproduces mainly by seeds. holm et al. (12) emphasized that due to the architecture of the fruit surface with large and small spines arranged at different angles with at least one of the spines always pointing upward. T. terrestris is capable of forming massive populations for a short period of time when the conditions are favorable (23).

the earliest presence of Tribulus terrestris in Bulgaria was shown by Velenovsky (28) in Flora Bulgarica (op.c.). later, Stojanov et al. (24) and Petrova (18) noticed that this species is widely distributed in Bulgaria up to an altitude of 800-1000 m. in consequence of their study on 56 localities and populations from South Bulgaria, Peev and Valyovska (16) defined T. terrestris as xeromesophylous. Up to now, in the world, in particular in our country, the investigations have referred mainly to the medicinal application of some active substances in this species. especially in Bulgaria, the over-ground part of the plant is used as a raw material for the medicine “tribestan”, which has a generally tonic action and stimulates some of the functions of the reproductive system.

this lM and SeM study was undertaken in order to reveal the pollen and seed morphology (the sporoderm and spermoderm repectively) of T. terrestris from Bulgaria, taking into account that generally in the world such important information for this species is rather vague and limited.

Materials and Methodsthe materials for the study were collected from a native Bulgarian population of T. terrestris from the thracian lowland, the valley of Karlovo, near to Vedrare village.

For the purpose of the pollen morphological study, dry pollen grains were used, after a light heating with an alcohol burner in a drop of acetocarmin on temporary slides. the shape, pollen diameter, polarity, symmetry of the pollen grains, and the features of sporoderm ornamentation were observed with lM “Amplival”. the photographs were taken with “Motik” DMBA 210. the diameter of the pollen grains, size of the brochi and thickness of the exine were observed at 16x40 magnification. Fifty measurements of 100 pollen grains were made and the obtained data were statistically analyzed.

the mathematical analysis of the data of all the pollen characteristics observed included: arithmetic average – ; average percentage error – S %; coefficient of variation – S%; max:min. For the pollen morphology the terminology and classification by Erdman (11) were used.

For the seed morphology of Tribulus terrestris the shape and sizes in mm, as well as the structure of the spermoderm were determined. In this case the terminology and classification by Barthlott and ehler (3) were used.

For SeM investigation the objects were directly observed, without any preliminary physical or chemical treatments. the seeds were observed in air-dry condition and the pollen grains were preserved in 95% ethanol. the scanning electron microscope JSeM - 5510 was used (the Faculty of chemistry, Sofia University) with gold coating in a vacuum-evaporator

POLLEN AND SEED MORPHOLOGY OF TRIBULUS TERRESTRIS L. (ZYGOPHYLLACEAE)

ivanka Semerdjieva1, elina Yankova-tsvetkova2, Georgi Baldjiev2 and Petka Yurukova-Grancharova2

1Agricultural University Plovdiv, Department of Botany, Plovdiv, Bulgaria2Institute of Biodiversity and Ecosystem Research, Bulgarian Academy of Sciences, Sofia, Bulgariacorrespondence to: ivanka Semerdgievae-mail: v_semerdjieva@abv.bg

ABSTRACTLight microscope (LM) and Scanning electron microscope (SEM) study of the pollen and seed morphology of Tribulus terrestris (Zygophyllaceae), especially in the native Bulgarian population from Thracian Lowland, was carried out. It was established that the pollen grains are oblate-spheroidal, radially symmetric, pantoporate. Exine ornamentation is reticulate. The seeds of Tribulus terrestris are flat, triangular-ovate. The seed sculpture corresponds to the Tubular-type, which is characterized with polygonal cells and straight arched anticlinal walls, whereas the periclinal ones are wavy with dense cuticular wax folds.

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for 60 seconds in an ionizing argon environment. the clamp holder for providing contact was made with silver paste. in the SeM study the method suggested by terziisky (25) was followed.

Results and DiscussionPollen morphologythe pollen morphological analysis has a proved taxonomical significance and is successfully used as an additional criterion for delimitation of the taxa, especially the polymorphous ones (26). on the basis of this analysis, el-hadidi (7, 8) excluded the genus Tribulus from the family Zygophyllaceae and placed it in a separate family Tribulaceae hadidi. other authors (14, 19), have placed this genus in the limits of the subfamily Tribuloideae (Rchb.) D.M. Porter. Using lM, teM and SeM, Praglowski (20) carried out a profound study on the sporoderm characteristics of some representatives of Tribulaceae. he noted that for the Tribulus genus the sexine is thicker than or as thick as the nexine and the endexine is very thin, fibrillar with very small globular elements.

the T. terrestris pollen grains studied with lM are spheroidal with a reticulate exine pattern (Fig. 1).

Fig. 1. General view of T. terrestris pollen grains (lM)

erdtman (11), Agababian (1), huang (13), Kuprianova and Aleyshina (15) described the pollen of this species as pantoporate, oblate-sheroidal, reticulate with different sizes of the brochi and a big lumen. Ben Mekki (4) observed a compact structure of the exine that is scaly inside. on the basis of lM and SeM study on the pollen morphology of Pakistan representatives of the Zygophyllaceae family, Perveen and qaiser (17) gave a key to the species of this family, in which four pollen types are determined: Nitraria retusa (Forssk.) Asch., Peganum harmala l., Tribulus terrestris and Zygophyllum simplex. these authors underlined that the Tribulus terrestris-pollen type holds in an isolated position, especially toward Nitraria retusa and Zygophyllum simplex l.-types. Perveen and qaiser (17) pointed out that in shape, the pollen grains of T. terrestris are spheroidal rarely oblate-spheroidal. the pollen of the studied population of T. terrestris is oblate-spheroidal and radially symmetrical (Fig. 2).

the sizes of the pollen grains range between 35 and 47.5 μm (43.5 μm at an average) (Table 1).

Fig. 2. Generall view of pollen grains (SeM)

TABLE 1Metric indexes of pollen grains in T. terrestris

Indexes `X S% S̀X% Max:MinDiameter in µm 43.16 7.28 1.32 1.35Size of brochi in µm 7.83 20.06 3.66 2.5thickness of exine in µm 5.06 12.62 2.3 1.5

in accordance with erdtman’s classification (11) they could be defined as pollen of a middle magnitude (ME). The exine ornamentation is reticulate with straight to slightly expressed wavy barriers (muri) with a simple columnar structure (Fig. 3).

Fig. 3. Structure of the pollen sexine (SeM)

The mean value of the exine size is 5.06 μm. Beug (6) has defined the pollen of T. terrestris as reticulate-porate with dimensions of the exine reticulate structures of 7-8 μm. The brochi are with a conically enlarged tip with a big lumen. the magnitude of the brochi is from 5 to 10.5 μm (7.83 μm at an

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average). the pores are disposed on the whole surface in the centre of each brochus, which gave us the reason to determine the pollen grains of T. terrestris as pantoporate. the shape of the pores is circular. Yunus and nair (29), Ben nasri-Ayachi and nabli (5), Perveen and qaiser (17) also determined the pollen grains of this species as pantoporate. the pantoporate pollen, which is a specific characteristic of T. terrestris, is an evidence of high evolution within the Zygophyllaceae family, according to Yunus and nair (29). it has been observed that the sexine of this species is thicker or thinner than that of the nexine (17, 29). in addition, Ben nasri-Ayachi and nabli (5), describing the pollen wall ultrastructure and its ontogeny in T. terrestris, underlined that the outer nexine is compact and the inner nexine is distinctly lamellar.

Seed morphologythe fruits are the basic source of information for identifying and classifying the taxa within the genus Tribulus (2). According to the diagnostic specific character of the fruits (mericarp peculiarites) in the genus Tribulus, el-hadidi (9) has determined three sections: 1. Terrestris which includes the species with spiny mericarps; 2. Alata - species with winged mericarps and section Inermis - species with spineless or wingless mericarps. he also recognized, just like engler (10), a fourth group with a section Terrestris including species whose mericarp is covered with several spines.

Until now, only scanty studies on the morphology of the seed coat sculpture in a small number of taxa from the Zygophyllaceae family have been carried out, especially conducted with SeM. they have been concentrated mainly on some species from the genera Fagonia l. and Zygophyllum (22).

Fig. 4.General view of a T. terrestris seed (SeM)

the seeds of T. terrestris are flattened, triangular-ovate with a sharp-lengthened tip and a flat base (Fig. 4, Fig. 5). the dimensions of the seeds are 3.5-4 mm. on the seed surface, wavy undulating folds were observed from its base to the top (Fig. 4, Fig. 5). At the apical part on the upper surface there is a three-ray split with various large and deep incisions (Fig. 6).

Fig. 5. General view of a seed (SeM)

Fig. 6. three-ray split on the seed surface (SeM)

laterally, longitudinal edges with deep concaves inside were observed in the two directions (Fig. 7).

Fig. 7. lateral longitudinal edge of the seed (SeM)

According to Barthlott and ehler (3), the seed surface could be described as tabular-type to slightly concave. From the analysis of our SeM study, it became clear that the cells of the spermoderm are polygonal in shape with straight to slightly arched anticlinal walls and with cuticular wax folds. the

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inner periclinal walls are wavy, slightly convex with densely interlaced cuticular striations (Fig. 8).

Fig. 8. General view of the seed spermoderm in T. terrestris (SeM)

As regards the morphology of the seeds, they are not such a useful source as the fruits for delimitation of the taxa within the genus Tribulus. Usually they are observed in connection with their role for effective reproduction of this species (2).

ConclusionsAs a result of this study, the morphological peculiarities of the pollen and seed of the valued medicinal plant Tribulus terrestris were determined using lM and SeM. it was observed that the pollen is pantoporate and mainly because of this feature T. terrestris occurred in a rather isolated position in the family Zygophyllaceae, in a particular subfamily Tribuloideae. Moreover, this type of pollen is considered as highly evolutionarily advanced within the family.

During the study, especially with SeM, it was established that the seed sculpture of T. terrestris referrs to tabular to slightly concave-type with polygonal cells of the spermoderm. these and other data obtained concerning the seed morphology could be considered as original for the species studied because such information is missing in the accessible literature sources.

Acknowledgments The financial support offered by the National Science Fund of Bulgaria (BnSF) (Ministry of education, Youth and Science) to the Project Do 02-246 is gratefully acknowledged. the authors thank Prof. DSc ilija cheshmedjiev for his help and advice.

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