Apocynaceae): an Evidence for Dual Cytokinesis in Microspore Mother Cells

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Microsporogenesis in Rauvolfia serpentina (L.) Benth ex Kurz (Apocynaceae): An evidence for dual cytokinesis in microspore mother cells

Balkrishna Ghimire1, Bimal Kumar Ghimire2 and Kweon Heo1*

1Department of Applied Plant Sciences, Kangwon National University, Chuncheon 200-701, South Korea. 2Department of Applied Life Science, Konkuk University, Seoul 143-701, South Korea.

Accepted 29 December, 2010

Rauvolfia serpentina is being used worldwide for its extensive medicinal value. A very few studies were done about microsporogenesis and male gametophyte formations in R. serpentina and they lack in accurate information about it. The aim of the present study was to give accurate information regarding microsporogenesis and male gametophyte formations in R. serpentina. Results explain the most important characteristic features of R. serpentina that was tetrasporangiate anther, dicotyledonous type of anther wall formation, the occurrence of both successive and simultaneous cytokinesis during the meiosis in microspore mother cells, uninucleate and highly vacuolated glandular tapetum, tetrahedral, tetragonal and decussate pollen tetrad and three-celled mature pollen grain during shedding time. These results were compared and discussed with other previous studies about Rauvolfia. Some of the studies contradicted with each other in terms of anther development and microsporogenesis, within genus and even in the species R. serpentina. Through this study, we clarified all these controversial information of microsporogenesis and concluded that R. serpentina undoubtedly follows both successive and simultaneous cytokinesis. Appearance of both types of cytokinesis is common in Rauvolfia, even though simultaneous is prevalent.

Key words: Rauvolfia serpentine, Apocynaceae, microsporogenesis, simultaneous cytokinesis, successive cytokinesis.

INTRODUCTION

Rauvolfia serpentina (L.) Benth. ex Kurz is an erect, has been recognized as antidotes to the stings and bites glabrous, perennial shrub, it is a good source of many of insects and poisonous reptiles in different parts of India important alkaloids of medicinal value (Alamgir and and Nepal (Vakil, 1955; IUCN Nepal, 2004). It has also Ahamed, 2005). It is one of the approximately known 80 been used as a febrifuge, as a stimulant to uterine species of Rauvolfia, which are mainly found in tropical contractions, for diarrhea, dysentery, insomnia and regions (Mabberley, 2008). Flowers borne in branched insanity (Vakil, 1955; Ahamed et al., 2002; IUCN Nepal, cyme and pentamerous except gynoecium, calyx 2004). synsepalous with imbricate lobes, corolla sympetalous The noteworthy contributions to the embryological forming a funnel shape with five limbs, and stamens are studies of the Apocynaceae were made by Fyre and epipetalous. Fruit are drupe, tiny, oval, fleshy which turn Blodgett (1905), Guignard (1917a,b), Anderson (1931), a shiny purple-black when ripe (Cronquist, 1981; IUCN Schnarf (1931), Meyer (1938) and Rau (1940). While Nepal, 2004). R. serpentina or common serpentine plant summarizing the embryological data on Apocynaceae, Davis (1966) mentioned the controversy concerning the nature of division of microspore mother cells and due to the occurrence of different types of embryogeny *Corresponding author. E-mail: [email protected]. Tel: 82- Apocynaceae termed as heterogeneous group by 33-250-6412. Fax: 82-33-244-6410. Maheshwari in 1971. Ghimire et al. 433

Microsporogenesis is the production of microspores from 0.1% Toludine blue O and stained slides were mounted with microspore mother cells in plants. The existing Entellan. Some flowers were also dehydrated by tertiary butyl alcohol

typological convention for microsporogenesis in angiosperms recognizes two patterns i.e. simultaneous series and embedded in paraplast of melting point 56 . Serial and successive, although intermediate patterns called sections of thickness 6 to 8 m were cut and dried on the slide modified simultaneous can also been seen (Sampson, warmer. Slides were stained with Heidenhain’s haematoxylin, safranin and fast green FCF and mounted with Entellan. All 1969a,b). The successive division involves the formation prepared slides were observed with the BX-50 light microscope of centrifugal cell plates, whereas simultaneous involves (Olympus Co, Japan). Photographs were taken by digital camera the formation of centripetal furrows, although this may not system attached to the microscope. be always the same case (Furness and Rudall, 1999). Early developmental events in microsporogenesis are known to play role in pollen morphology and tetrad shape. RESULTS Successive cytokinesis, particularly common in monocots (Rudall et al., 1997; Furness and Rudall, 1999) generally The flowers are bisexual, pink pedicels and calyx, white leads to tetragonal, decussate, T-shaped or linear tetrad. corolla, and inflorescence irregular corymbose cymes Simultaneous cytokinesis, on the other hand, is mostly (Figure 1). Floral parts are arranged in pentamerous associated with tetrahedral tetrad. This type is found in except gynoecium (Figure 2B). Calyx synsepalous with monocots, but is the rule in eudicots (Rudall et al., 1997; imbricate lobes and persistent, corolla sympetalous Furness and Rudall, 1999; Furness et al., 2002). Majority forming a funnel with five limbs which will be deciduous of genera in Apocynaceae follow simultaneous pattern of after fertilization and stamens are epipetalous (Figures 1 microsporogenesis (Davis, 1966; Johri et al., 1992). and 2A-B). The ovary was superior, bicarpellary and However, successive cytokinesis occurs in Apocynum bilocular. Each stamen appears basifixed and androsaefolium, Holarrhena antidysentrica (Lattoo, 1974), tetrasporangiate anther (Figures 2A-B). Young flowers Plumeria diffusa and Plumeria rubra (Chauhan, 1979). has very short style and stamen around the carpel Simultaneous as well as successive cytokinesis has been (Figures 2A-B), but in open flowers, style extends its reported in Catharanthus pusillus (Bhasin, 1971) and length and corolla grows into a long tube. Therefore, Rauvolfia canescene (Meyer, 1938). Maheshwari (1970) stamen is located in upper part of corolla tube. and Lamba (1976) mentioned only simultaneous cytokinesis in R. serpentina and same for Rauvolfia sumatrana by Lakshminarayana (1988). As mentioned above single genus Rauvolfia has been confirmed with Anther wall formation diverse mode of microsporogensis. In the same way, Bhasin (1971) and Lamba (1976) reported two-celled The anther wall, prior to maturity, was four to five layered: pollen in Catharanthus pusillus and R. serpentina an epidermis, an endothecium, one or two middle layers respectively but three-celled pollen grains had been and a tapetum (Figure 3A-D). The primary parietal cell reported in R. serpentina (Maheshwari, 1970) and in R. undergoes periclinal and anticlinal divisions and forms sumatrana (Lakshminarayana, 1988). secondary parietal layer. The secondary parietal layer These inaccurate and contradicting information about adjacent to epidermis again divides and produces microsporogenesis and number of cells in mature pollen endothecium and middle layers (Figure 3A). The middle during shedding in genus Rauvolfia facilitate us to re- layer further divides periclinally and becomes two layered examine the microsprorgenesis and anther characters of structure (Figure 3B). The secondary parietal layer R. serpentina. The purpose of this study was to present adjacent to sporogenous cells functions as tapetum truthful information about microsporogenesis of R. without further division (Figure 3A-B). Thus, the anther serpentina and to produce a comprehensive wall formation is dicotyledonous type in which only the understanding about microsporogenesis and other anther outer secondary parietal layer takes part in the formation characteristics in Rauvolfia. of middle layer (Figure 3A). Glandular tapetum surrounds sporogenous tissue (Figure 3C). The tapetal cells remain uninucleate throughout and some of them undergo a MATERIALS AND METHODS periclinal division resulting in a two-layered condition (Figure 3C). Their dense cytoplasm become vacuolated Samples ranging from small buds to open flowers of by the time tetrad are formed (Figure 3C). The glandular differentdevelopmental stages were collected and fixed in FAA (5 tapetum degenerate only after the pollen reaches the parts formalin: 5 parts glacial acetic acid: 90 parts 50% ethanol) for two-celled stage. The middle layer is ephemeral and one week and then preserved in 50% ethanol. Preserved buds and degenerates soon while the endothecium develops flowers were dehydrated by ethanol series (50, 70, 80, 90 and 95% and absolute ethanol). After dehydration, buds and flowers were fibrous thickenings (Figure 4F-G). The anther epidermal embedded in Technovite 7100 resin and histo blocks were cells are nearly collapsed at the time of pollen shedding. prepared. Serial sections of thickness 4 to 5 m were cut by rotary Thus, the mature anther wall is composed of a sole microtome using a disposable knife. Staining was performed by fibrous endothecium layer (Figure 4G). 434 J. Med. Plants Res.

Figure 1. Inflorescence and flowers of Rauvolfia serpentina. Scale bar indicates 3 cm.

Microsporogenesis and male gametophyte 3J-L). On the other hand, no cell plate was laid down after meiosis I, and the spindle fibers of this division The primary sporogenous cells directly function as disappear during the metaphase of meiosis II (Figure 4A). microspore mother cells. The microspore mother cells After the four daughter nuclei have become organized, undergo two consequent meiosis divisions and forms they assumed a tetrahedral arrangement and spindle was microspore tetrad. Cell division of microspore mother cell reformed between every two nuclei (Figure 4B). It is begins with early prophase I and ends with late telophase followed by formation of constriction furrows which start II passing through metaphase I, anaphase I, telophase I, at the periphery and proceed inward until they meet at prophase II, metaphase II and anaphase II (Figures 3E-L the center, so that there is simultaneous division of and 4A-D). During meiosis II, the spindles oriented protoplast into four cells microspores (Figure 4C). The parallel or right angles to each other (Figure 4A) forming resultant microspores arranged themselves in tetrahedral, microspore tetrads (Figure 4D). Cytokinesis in tetragonal and decussate fashion (Figure 4D). The microspores follows both successive (Figures 3J-K) and microspores soon separated from each other and simultaneous (Figure 4B-C) types although later one was individually developed into pollen grains. The mature more frequent than former. In successive cytokinesis, cell pollen grains are triporate surrounded by exine and intine plate was laid down immediately after the telophase of (Figure 4H) and they are three-celled, one large first meiotic division and another in each of the two vegetative cell and a central generative cell with two daughter cells after the second meiotic division (Figure nuclei during shedding (Figure 4I). The dehiscence of Ghimire et al. 435

Figure 2. Young flower of R. serpentina. (A) Longitudinal section of young flower showing sepal, petal, stamens and carpel (arrow indicates the epipetalous stamen). (B) Transverse section of young flower with different parts arranged in whorl. Abbreviations: ca, carpel; pe, petal; se, sepal; st, stamen. Scale bars indicate 100 µm in Figure 2A and 50 µm in Figure 2B.

Figure 3. Anther wall formation and meiosis I in microspores: (A) Transverse section of young anther of R. serpentina showing dicotyledonous type of wall formation. (B) Anther wall showing periclinal division of middle layer. (C) Uninucleate tapetal cells with large vacuole. (D) Five-layer anther wall with glandular tapetum. (E-F) Prophase I in microspore mother cell. (G) Metaphase I. H- I Anaphase I early and late, respectively. J Telophase I. K-L Successive cytokinesis in microspore. Abbreviations: et, endothelium; ep, epidermis; ml, middle layer; pmc, pollen mother cell; t, tapetum. Scale bars indicate 10 µm in all figures. 436 J. Med. Plants Res.

Figure 4. Microsporogenesis and mature pollen of R. serpentina. (A) Metaphase II. (B) Telophase II. (C) Simultaneous cytokinesis. (D) Pollen tetrads. (E) Mature anther wall with fibrous endothecium. (F-G) Longitudinal slits of mature anther. (H) Mature tricolpate pollen. I Three-celled pollen. Abbreviation: ep, epidermis; et, endothecium; gc, generative cell; vc, vegetative cell. Scale bars indicate 10 µm in Figure 4A-E, 50 µm in Figure 4F-G, and 20 µm in Figure 4H-I.

anther takes place through a longitudinal slit (Figure 4F). Comparisons within apocynaceae Firstly, suture of between two microsporangia in an anther lobe detaches, soon after pollens released from The majority of the anther and microspore characteristics microsporangium through a common slit formed in observed in R. serpentina are coincides with general between two microsporangia of the same lobe (Figure features of Apocynaceae. Tetrasporangiate anther found 4G). in R. serpentina is also found in most of the Apocynaceae. However, Sud (1984) confirmed bisporangiate anther in Trachelospermum fragrans. The development of anther DISCUSSION wall in R. serpentina is obviously dicotyledonous type (Lakshminarayana, 1988) and prior to maturity it is A summary of anther and microsporogenesis of R. multilayered, a single middle layer later divides to two. serpentina Precisely same pattern has been seen in Holarrhena antidysenterica (Lattoo, 1974), Vallaris heynei (Rau, Previous studies about Rauvolfia lacked in terms of 1940) and 2-3 middle layers reported in T. fragrans (Sud, detailed about microsporogenesis and anther wall 1984). This is contrary to Maheshwari (1971) who formation (Maheshwari, 1970; Lamba, 1976; reported a 10-14 middle layers in V. foetida. Some cells Lakshminarayan, 1988). Present study has clarified many of the tapetum also divide, and at places it becomes two- distinguished characteristics features of anther and layered. Such a condition has also been reported by microsporogenesis, like anther tetrasporangiate; anther Lattoo (1974) in H. antidysenterica and by Maheswari wall prior to maturation consists up to five cell layers; (1964) in species of subfamily Asclepiadoideae (formerly anther wall formation dicotyledonous type; anther known as a separate family Asclepiadeceae) for example epidermis nearly collapsed; middle layer degenerated; Cynanchum callialata, Pergularia daemia and Tylophora tapetum glandular, usually tapetal cells uninucleate; indica. The most important characteristic in R. serpentina cytokinesis in microspore mother cell follows both found in this study was both successive and successive and simultaneous types; microspore tetrads simultaneous cytokinesis during the meiosis of pollen are tetrahedral, tetragonal and decussate in shape; mother cells. Similar characteristics can be seen in other pollen grains are triporate, three-celled during shedding Apocynaceae members like, Catharanthus pusillus time and anther dehiscence by longitudinal slit. (Bhasin, 1971) and R. canescence (Meyer, 1938). But Ghimire et al. 437

previous studies in R. serpentina (Maheshwari, 1970; cytokinesis, even though simultaneous is predominant Lamba, 1976) and R. sumatrana (Lakshminarayana, and pollen grain shed at three celled stage. Through this 1988) described only simultaneous cytokinesis which study we clarified all these controversial information on should be revised at this time. Only simultaneous microsporogenesis and pollen grain concerning to genus cytokinesis in microspore mother cells has been reported Rauvolfia. As we might articulate from previous in T. fragrans (Sud, 1984) whereas only successive information and this study microsporogenesis is highly cytokinesis has been found in H. antidysenterica (Lattoo, diverse in family Apocynaceae. However, further studies 1974). Apart from Apocyanaceae both successive and are required on other genera of Apocynaceae before to simultaneous cytokinesis in a same species has been make any comment on definite evolutionary significance observed in Vernonia cinerea (Asteraceae) by Dakshini and diversity of microsporogenesis. and Dadlani (1978) and in one cultivar of Codiaeum variegatum var. pictum (Euphorbiaceae) by Albert et al. (2009). It is rare for a species to have both types of ACKNOWLEDGEMENTS cytokinesis and specially highly unusual for eudicots species, since in higher eudicots, cytokinesis is uniformly The authors appreciate the Bio-herb research center of described as simultaneous (Longly and Waterkeyn, 1979; Kangwon National University for supporting experimental Blackmore and Barnes, 1988; Otegui and Staehelin, instruments. We also thank two anonymous reviewers for 2000; Ressayre et al., 2000) with few exceptions e.g. valuable comments. Porteaceae (Blackmore and Barnes, 1995) Rafflesia (Rafflesiaceae) (Furness and Rudall, 2004) and REFERENCES Podostemaceae (Furness and Rudall, 2004; Jager-Zurn et al., 2006). In contrast to eudicots, cytokinesis is highly Ahmad S, Amin MN, Mosaddik MA (2002). Studies on moisture content, labile in basal angiosperms and monocots (Furness and biomass yield (crude plant extract) and alkaloid estimation of in-vitro Rudall, 1999; Furness et al., 2002). In R. serpentina and field grown plants of Rauvolfia serpentina. Pakistan. J. Biol. Sci., 5: 416-418. pollen grains are shed at the three-celled stage like as in Alamgir ANM, Ahamed M (2005). Growth and phytochemical most other Apocynaceae. However, anthesis at two- investigation of Rauvolfia serpentina Benth. Bangladesh J. Bot., 34: celled stage has been reported by Bhasin (1971) in C. 7-10. pusillus and by Lamba (1976) in R. serpentina. Present Albert B, Gouyon PH, Ressayre A (2009). Microsporogenesis variation in Codiaeum producing inaperturate pollen grain. C. R. Biologies, study results confirm Lamba’s (1976) study on same 332: 507-516. species contradict in two important embryological Anderson A (1931). Studies on the embryology of the family features, that is, cytokinesis of microspores and number Celastraceae, Oleaceae and Apocynaceae. Acta Univ. Lund., 27: 1- of cells in mature pollen grains. Three-celled pollen 112. Bhasin RK (1971). Studies in Apocynaceae. II. A contribution to the grains feature had already been reported in R. serpentina embryology of Catharanthus pusillus (Murr.) G. Don. Botanique (Maheshwari, 1970) which is confirmed in R. sumatrana (Nagpur, India), 2: 111-122. (Lakshminarayana, 1988) and R. serpentina (this study). Blackmore S, Barnes S (1988). Pollen ontogeny in Catananche In general, the mode of cytokinesis, shape of tetrad and caerulea L. (Compositae, Lactuceae). 1. Premeiotic phase to establishment of tetrads. Ann. Bot., 62: 605-614. number of cells in mature pollen are considered as the Blackmore S, Barnes SH (1995). Graside’s rule and the microspore most consistent characters in generic level, in some tetrads of Grevillea rosmarinifolia A. Cunningham and Dryandra cases within the family level as well (Tobe, 1989). Within polycephala Bentham (Protaceae). Rev. Palaeobot. Palynol., 85: monocotyledon microsporogenesis is a significant 111-121. Chauhan TS (1979). Morphological studies in Apocynaceae. II. character at the ordinal level and is usually (but not Embryology of Plumeria. J. Indian Bot. Soci., 58: 363-368. always) consistent within a family (Furness and Rudall, Cronquist A (1981). An integrated system of classification of flowering 1999; 2000). But surprisingly, earlier reports on these plants. Columbia University Press, New York. characters in genus Rauvolfia vary with each other Dakshini KMM, Dadlani NP (1978). Unusual diversity in microsporogenesis in Vernonia cinerea. Bot. Mag. Tokyo, 91: 131- (Maheshwari, 1970; Lamba, 1976; Lakshminarayana, 135. 1988) even within same species R. serpentina Davis GL (1966). Systematic embryology of angiosperms. John Wiley (Maheshwari, 1970; Lamba, 1976). However, from this Co. Press, New York. study we are clear about these two features in R. Furness CA, Rudall PJ (1999). Microsporogenesis in monocotyledons. Ann. Bot., 84: 475-499. serpentina that: cytokinesis followed by both types which Furness CA, Rudall PJ (2000). The systematic significance of has also been reported for another species R. simultaneous cytokinesis during microsporogenesis in canescence by Meyer (1938) and pollen grain released at monocotyledons. In: Wilson Kl, Morrison DA (eds) Monocots: three celled stage also reported by Maheshwari (1970) Systematic and Evolution Vol 1: Proceedings of the second international conference on the comparative biology of the monocot, for R. serpentina and by Lakshminarayana (1988) for the CSIRO, Melbourne, pp. 186-190. R. sumatrana. Furness CA, Rudall PJ (2004). Pollen aperture evolution – a crucial In conclusion our result is more consistent and clear factor for eudicot success? Trends Plant Sci., 9: 154-158. about the most of anther characteristics of R. serpentina. Furness CA, Rudall PJ, Sampson FB (2002). Evolution of microsporogenesis in angiosperms. Int. J. Plant Sci., 163: 235-260. R. serpentine undoubtedly follows both type of Fyre TC, Blodgett EB (1905). A contribution to the life history of 438 J. Med. Plants Res.

Apocynum androsamifolium. Bot. Gaz., 40: 49-53. Rau MA (1940). Studies in Apocynaceae. J. Indian Bot. Soci., 19: 33-44. Guignard L (1917a). The development and structure of the egg in Ressayre A, Raquin C, Mignot A, Godelle B, Gouyon PH (2002). Apocynaceae and Asclepiadaceae. The Proceedings of the Acad. Correlated variation in microtubule distribution, callose deposition Sci., (Paris), 165: 981-987. during male post-meiotic cytokinesis, and pollen aperture number Guignard L (1917b). The egg in Apocynaceae and Asclepiadaceae. across Nicotiana species (Solanaceae). Am. J. Bot., 89: 393-400. Memories of the Academy Sci. Insti. France, 55: 1-34. Rudall PJ, Furness CA, Chase MW, Fay MF (1997). Microsporogenesis IUCN Nepal (2004). National register of medicinal and aromatic plants and pollen sulcus type in Asparagales (Lilianae). Can. J. Bot., 75: (revised and updated). IUCN - The world conservation union, Nepal. 408-430. Jager-Zurn I, Novelo A, Philbrick CT (2006). Microspore development in Sampson FB (1969a). Cytokinesis in pollen mother cells of Podostemaceae-Podostemoideae, with implications on the angiosperms with emphasis on Laurelia novae-zelandiae characterization of the subfamilies. Plant Syst. Evol., 256: 209-216. (Monimiaceae). Cytol., 34: 627-634. Johri BM, Ambegaokar KB, Srivastava PS (1992). Comparative Sampson FB (1969b). Studies on the Monimiaceae. I. Floral embryology of angiosperms. Springer-verlag Press, Berlin, morphology and gametophyte development of Hedycarya arborea J. Heidelberg. R. et G. Forst. (sub-family Monimioideae). Austr. J. Bot., 17: 403-424. Lakshminarayana K (1988). Male and female gametophyte of Rauvolfia Schnarf K (1931). Vergleichende embryologie der angiospermen. sumatrana Jack. Curr. Sci., 57: 85-87. Borntraeger, Berlin. Lamba LC (1976). Microsporogenesis and male gametophyte in Sud KC (1984). A contribution to the embryology of Trachelospermum Rauvolfia serpentina (L.) Benth. ex. Kurz. Curr. Sci., 45: 387-388. fragrans Hook. F. (Apocynaceae). Proceeding of Indian Acad. Sci. Lattoo CS (1974). Morphology and embryology of Holarrhena (Plant Science), 93: 495-501. antidysenterica Wall. Bot. Gaz., 135: 173-180. Tobe H (1989). The embryology of angiosperms: its broad application to Longly B, Waterkeyn L (1979). Etude de la cytocinese II. Structure et the systematic and evolutionary study. Bot. Mag. Tokyo, 102: 351- isolement des plaques cellulaires microsporocytaires. La Cellule, 72: 367. 227-242. Vakil RJ (1955). Rauvolfia serpentina in the treatment of high blood Mabberley DJ (2008). A portable dictionary of plants, their classification pressure. Circulation, XII: 220-229. and uses. Cambridge University Press, Oxford. Maheswari DH (1964). Embryological studies in Asclepadaceae. Proc. Indian Acad. Sci., 60: 52-65. Maheswari DH (1970). Embryology of Rauvolfia serpentina and Catharanthus roseus. Curr. Sci., 39: 376-377. Maheswari DH (1971). Embryology of Apocynaceae. I. Plumiereae. J. Indian Bot. Soci., 50: 74-85. Meyer S (1938). Studies in the family Apocynaceae. J. Depart. Sci. Calcutta University, 1: 131-158. Otegui M, Staehelin LA (2000). Cytokinesis in flowering plants: more than one way to divide a cell. Curr. Opin. Plant Biol., 3: 493-502.