Analele ştiinţifice ale Universităţii Al. I. Cuza Iaşi Tomul LIV, fasc. 2, s.ii a. Biologie vegetală, 2008 MICROSPOROGENESIS AND THE MALE GAMETOPHYTE AT EPHEDRA DISTACHYA L. GENŢIANA MIHAELA IULIA PREDAN *, IRINA GOSTIN ** Abstract: This study describes the microsporogenesis and the male gametophyte in Ephedra distachya L., only one species of the Ephedraceae family growing spontaneous in Romania. Ephedra distachya L. is a dioecious plant. Light microscopy (lm) is used to examine the early developmental stages of the male cones and combined light (lm) and scanning electron microscopy (sem) were used to analyze the mature pollen grains. The pollen cones are clustered at nodes either sessile or stalked. They are oval-oblong, each is composed of 2-8 decussate pairs of membranous bracts, proximal bracts empty; each distal bract subtending a male flower composed of 2 basally fused, orbicular or obovate scales (false perianth); sessile anthers on staminal column. Ephedra distachya pollen is particularly abundant. Ephedra distachya has the polyplicate pollen type characterized by a pointed-oval shape and 6 longitudinal ridges and 6 valleys, due to alternation of thicker and thinner exine regions, inaperturate. In polar view, ridges are low and triangular. On the SEM analysis the pollen wall of Ephedra distachya is simple, so the SEM closely resembles the LM observation. Key words: Ephedra distachya L., male cones, pollen development Introduction Genus Ephedra L. contains 35-45 species, most of them populating the desert or arid regions. In Romania a single species of Ephedraceae family occurs - Ephedra distachya L. Its status is rare [12]. Genus Ephedra L. has been much studied from the morphological and anatomical point of view, information was summarized and included in synthesis works [8, 10, 14, 15, 16]. The histology of the flowers and strobili of the Ephedra distachya L. plants was examined by Van Tieghem (1869), Thoday and Berridge (1912) [10], later by Favre-Duchartre M. [5]. Baranec T. Rehorek and V. studied the reproductive cycle of the Ephedra distachya L. plants, spontaneous in Slovakia [2] and P. Mehra N. analysed dimensions of the male nuclei of the representatives in the Ephedra genus [10]. Allison S. D. et al. have studied all the important stages of the Ephedra pollen development using both photonic and the scanning and transmission electronic microscope [3]. Gamal El-Ghazaly et al. were concerned about pollen grain polarity, the aperture situation and the pollen tube at 5 species of Ephedra including E. distachya L. [6]. The microsporogenesis and the description of the pollen and the male gametophyte * University of Bucharest, Faculty of Biology, Department de Botany and Microbiology, Aleea Portocalelor, nr. 1-3, 060101, Bucureşti, Romania; ggentiana@yahoo.com ** Al. I. Cuza University, Faculty of Biology, Bd. Carol I, no. 20A, 700506, Iaşi, Romania; irinagostin@yahoo.com 24
development in Ephedra distachya L. provides interesting characterization in this species. Material and methods Our study was carried out on samples of Ephedra distachya L. from different places in Romania: Agigea Marine Dune Reserve (Constanta County), Culmea Pricopanului (Tulcea County), the Botanical Garden of Iaşi (cultivated plants) and from Bulgaria (Balchik) [7, 8]. Ephedra distachya L. is a dioecious plant. Male cones were collected during the three spring months (March, April and May), in successive phaenological phases, from the cones initiation up to the pollen shedding. The biological material (male cones) was fixed in 70% alcohol or FAA (formalin 40%, 5 cc: alcohol 70%, 90 cc: glacial acetic acid 5 cc). After being fixed, the material has been processed in accordance with the paraffin embeding protocol [3], sectioned (transverselly and / or longitudinally) with a Minot type microtome at 12-15 μm thick, colored with Ehrlich s haematoxylin and embedded in Canada balm. To highlight microspores and young pollen grains microscopical preparations squash type have been made, that have been colored with acetic carmine. All obtained sections were analyzed using light microscope (LM) Docuval type and photographed with a digital photo camera slr Canon EOS 400D. For the analysis of the pollen grains at electronic scanning microscope (SEM) a Vega - TESCAN LSH microscope was used in The Faculty of Biology of The Al I. Cuza University from Iaşi [12]. Male cones were considered from the morphological point of view with a Belphotonics stereomicroscope and photographed with the same digital camera. The description of the pollen grains has been made in accordance with the terminology adopted by Faegri [4]. Results and discussions The male cones, either sessile or stalked, are found clustered at nodes (fig. 1). The male cones are green, oval-oblongue, each of them being composed of 2-8 descussate pairs of membranous bracts, like cups (Fig. 2, 3). The lower pair bracts is sterile, each distal bract subtending a male flower composed of 2 basally fused, orbicular or obovate scales (primitive periant) (Fig. 4, 7), an axis (microsporangiophore or anterophore) terminal bearing a number of anthers, each anther with two micro sporangia (pollen sacs) (Fig. 4). A group of archespores subepidermal cells in the mature cones is observed in March (fig. 5). In April, following meiosis I, macrospore dyads have resulted. After meiosis II numerous tetrads of microspores appear, the wall formation taking place after each stage of meiosis. Soon, the wall surrounding the 4 microspore cells dissolve and the microspores in the pollen sac are 25
released. The microsporangia wall is composed of three known layers: epidermis, the median layer and the tapetum layer. The median layer is composed of a single row of parenchimatic cells and, as the pollen sac grows, its cells are flattened so that they ultimately disappear. The tapetum has signs of degeneration after the reductional division (Fig. 6) and no longer in the stage of mature pollen (Fig. 8). At mature pollen sac epidermis persists. Its cells have a regular appearance and their walls are thickened (Fig. 9). Only in a small portion of the pollen sac upper part a few parenchimatic cells with thin walls remain. By their breaking a hole will form that will be issued pollen (poricide opening). In May, the pollen is divided and forms a small prothalian cell and a large, central one (Fig. 10). The last divide and formed the second prothalian cell and initial anteridial cell. The last divide and give a vegetative larger cell, and a generative cell. The generative cell will form the stalk cell and spermatogene cell, the latter giving rise to gametes. Regarding pollen sacs, the pollen has 5 cells [8]. The mature pollen grains are yellow, fusiforme and obvious poliplicate: they have 6 longitudinal ridges (plicae) (sometimes slightly corrugated) and 6 valleys, formed due to the alternation of thicker and thinner exine regions (Fig. 9, 10). On a polar view, the ridges are short and triangular. Pollen grains have 53 μm long axis and 20 μm short axis. At the electronic scanning microscope (SEM), the pollen surface is no obvious regulate and no aperture was noticed eather, so the pollen is inaperturate (Fig. 11, 12). Conclusions In March, a group of archespore cells appears in the pollen sacs, subepidermaly. In April the anther wall is composed of an epidermis and a scrap tapetum and the microspores are free. In May the anther wall is only represented by the epidermis and numerous grains of fusiforme and yellow pollen are found inside the pollen sac, in our observation (in 17 May 2007 on Culmea Pricopanului) the pollen grains are uninucleate. According to the SEM observations, the pollen grains of Ephedra distachya L. have a simple structure, the SEM observations being very similar to those made on an optical microscope. REFERENCES 1. ANDREI M., RĂDULESCU D., 1972 - Caiet pentru tehnica preparării şi conservării materialului biologic. Tipogr. Univ. din Bucureşti 2. BARANEC, T., V. REHOREK, et al., 1994 - Generative reproduction of ephedra (Ephedra distachya L.) in Slovakia. Biologia Bratislava, 49 (1): 65-67 3. DOORES ALLISON S., OSBORN J. M., GAMAL EL-GHAZALY., 2007 - Pollen Ontogeny in Ephedra americana (Gnetales). International Journal of Plant Sciences 168 (7): 985-997 4. FAEGRI K., IVERSEN J., 1966 - Textbook of pollen analysis. 2nd Ed. Munksgaard, Copenhagen 26
5. FAVRE-DUCHARTRE M., 1959 - Contribution à l étude de la reproduction sexuée chez Ephedra distachya. C. R. Acad. Sci. Paris. 249: 1551-1553 6. GAMAL EL-GHAZALY, ROWLEY J., HESSE M., 1998 - Polarity, aperture condition and germination in pollen grains of Ephedra (Gnetales). Plant Systematics and Evolution, 213 (3-4): 217-231 7. GRINŢESCU G. P., 1952 - Ephedra L. In: T. SĂVULESCU (red. princip.). Flora României. 1. Bucureşti: Edit. Academiei Române 8. JOHRY B. M., BISWAS C., 1997 - The Gymnosperms. Narosa Publishing House, New Delhi 9. KRÜSSMANN G., 1985 - Manual of cultivated conifers. B.T. Batsford Ltd. London 10. LEHMANN-BAERTS M., 1967 - La morphologie du sporophyte dans le genre Ephedra. La Cellule (Belg.), 67 (5): 7-56 11. MEHRA P. N., 1950 - Inequality in size of the male nuclei in the genus Ephedra. Ann. Bot., July 1950; 14: 331-339 12. OLTEAN M., NEGREAN G., POPESCU A., ROMAN N., DIHORU G. SANDA V., MIHĂILESCU S., 1994 - Lista roşie a plantelor superioare din România. Studii, sinteze, documentaţii de ecologie, 1 13. PLOAIE G. P., PETRE ZOE. 1979 - Introducere in microscopia electronica cu aplicatii in biologia celulara si moleculara. Edit. Acad. Romane, Bucuresti 14. SCHNARF K., 1933 - Embryologie der Gymnospermen. In: Linsbauer K. Handbuch der Pflanzenanatomie, II Abt., 2 Teil, Bd. 2. Verlag Gebruder Borntraeger, Berlin 15. SINGH H., 1978. - Embryology of gymnosperms. Berlin-Stuttgart 16. SMARANDACHE D., 2005 - Embriologia plantelor. I. Embriologia arhegoniatelor. Edit. Univ. din Bucureşti, Bucureşti. 17. STEEVES M. W., BARGHOORN E. S., 1959 - The pollen of Ephedra. J. Arnold Arboretum, 40: 221-255 The explanation of figures: Plate I Fig. 1 Ephedra distachya L. - Branch with male cones (1 May 2008; Orig) Fig. 2 Ephedra distachya L. Male cone (20 May 2007) (Oc. 10x; Amplific. 2; Orig.) Fig. 3. Ephedra distachya L. - Longitudinal section of a male cone - fragment (13 April 2008) (Oc 12.5x; ob. 3.2x; Amplific. 8; Orig.) Fig. 4. Ephedra distachya L. - Male flower (20 May 2007) (Oc. 10x; Amplific. 3; Orig.) Fig. 5. Ephedra distachya L. - Longitudinal section through the anther that outlines the archaesporal cells (17 March 2007) (Oc 12.5x.; ob. 25x; Amplific. 160; Orig.) Fig. 6. Ephedra distachya L. - Cross-section through the male flower (13 April 2008) (Oc. 12.5x; ob. 10x; Amplific. 6.3; Orig.) Fig. 7. Ephedra distachya L. - Longitudinal section through the male flower (13 April 2008) (Oc. 12.5x; ob. 10x; Amplific. 6.3; Orig.) Plate II Fig. 8 Ephedra distachya L. - Longitudinal section through the male flower (29 May 2001) (Oc. 12.5x; ob. 3.2x; Amplific.16; Orig.) Fig. 9. Ephedra distachya L. - Longitudinal section through the anther - anteral wall and young pollen grains (29 May 2001) (Oc. 12.5x; ob. 40x; Ampific. 16; Orig.) Fig. 10. Ephedra distachya L. - Unicellular pollen grain (17 May 2007) (Oc. 12.5x; ob. 40x; Amplific.16; Orig.) Fig. 11. SEM micrograph of Ephedra distachya L. poliplicate pollen (21 May 2008; Orig.) Fig 12. SEM micrograph of Ephedra distachya L. pollen (21 May 2008; Orig.) 27
GENŢIANA PREDAN, IRINA GOSTIN PLATE I 1 2 3 4 5 6 7 28
GENŢIANA PREDAN, IRINA GOSTIN PLATE II 8 9 10 11 12 29