- Email: [email protected]
Androecium development in the bromeliad Dyckia pseudococcinea L.B.Sm. (Pitcairnioideae-Bromeliaceae), an endangered species endemic to Brazil: implications for conservation
Flora 207 (2012) 622–627
Contents lists available at SciVerse ScienceDirect
Flora journal homepage: www.elsevier.de/flora
Androecium development in the bromeliad Dyckia pseudococcinea L.B.Sm. (Pitcairnioideae-Bromeliaceae), an endangered species endemic to Brazil: implications for conservation Simone Petrucci Mendes a,∗ , Cecília G. Costa b , Karen L.G. De Toni b a b
Museu Nacional/Universidade Federal do Rio de Janeiro, Quinta da Boa Vista s.n., São Cristovão, Rio de Janeiro, RJ, Brazil Instituto de Pesquisas Jardim Botânico do Rio de Janeiro, Diretoria de Pesquisa, Pacheco Leão 915, Jardim Botânico, Rio de Janeiro, RJ, Brazil
a r t i c l e
i n f o
Article history: Received 9 December 2011 Received in revised form 5 May 2012 Accepted 26 May 2012 Keywords: Anther Tapetum Androsporogenesis Androgametogenesis Pollen grains Conservation
a b s t r a c t Dyckia pseudococcinea L.B.Sm. is endemic to the restingas of Maricá, State of Rio de Janeiro, in southeastern Brazil. However, since this area is under intense ecological stress, D. pseudococcinea is considered an endangered plant species. An important step toward the conservation of this species is establishing the developmental stage most favorable to in vitro propagation. Accordingly, the present study analyzed the androecium to establish the developmental stages of anthers, emphasizing anther wall development, androsporogenesis and androgametogenesis. The relationships between the size of flowers and anthers were also studied. Anther wall development follows the basic-type, while the tapetum, which is originated from the subepidermal layer (inner secondary parietal layer 2 and archesporial initials), follows the secretory-type. Androsporogenesis is successive and originates isobilateral and decussate tetrads. The oblate and monosulcate pollen grains exhibit microreticulate exine and are dispersed at the bicellular stage. At the vacuolated stage, the androspore (microspore) is still proliferating and not yet completely differentiated. Therefore, since the androspore has not yet lost its embryonic capacity, we suggest the preferential use of this developmental stage, which is present in flower buds 6.4–7.1 mm in length, in conservation protocols focused on the androgenesis of D. pseudococcinea. © 2012 Elsevier GmbH. All rights reserved.
Introduction Bromeliaceae is one of the most common families in restinga ecosystems of the Brazilian Atlantic Forest (Mantovani and Iglesias, 2001), with more than one third of the species being endemic to this biome (Martinelli et al., 2008). However, only 12% of the forest original area remains, mainly because of uncontrolled heavy exploration for commercial purposes and the associated environmental degradation (Ribeiro et al., 2009). This has strongly contributed to the dramatic reduction, or even extinction, of many species, also for the Bromeliaceae. Therefore, rare and endemic species are now frequently targets of conservation initiatives, since they are particularly vulnerable and prone to the risk of extinction (Scarano, 2009), in some cases being also keystone species or flagship taxa that sensitize the public for the ecological deterioration. Dyckia pseudococcinea L.B.Sm. belongs to such taxa and is included in the Brazilian List of Threatened Plant Species (MMA, 2008), mainly because of
∗ Corresponding author at: Instituto de Pesquisas Jardim Botânico do Rio de Janeiro, Diretoria de Pesquisa, Rua Pacheco Leão 915/sala 306, CEP 22460-030, Jardim Botânico, Rio de Janeiro, RJ, Brazil. Tel.: +55 21 3204 2099. E-mail address: [email protected] (S.P. Mendes). 0367-2530/$ – see front matter © 2012 Elsevier GmbH. All rights reserved. http://dx.doi.org/10.1016/j.flora.2012.06.016
its endemism to the restingas of Maricá, State of Rio de Janeiro, in southeastern Brazil, an area under intense ecological stress. Aimed at species conservation, in vitro culture of anthers has been broadly used in the production of haploid and double haploid plants for reintroduction in degraded areas (Hoffmann et al., 1982; Siebel and Pauls, 1989). It is considered a means to avoid ultimate extinction of plant species, albeit at a low level of genetic diversity. However, correctly identifying the stages of anther development is critical to the success of in vitro cultures (Gribaudo et al., 2004; Vidal et al., 2009). As such, understanding the ontogenetic details of anther development is a precondition in propagation studies. At the same time, however, evolutionary analysis in angiosperms depends generally upon an understanding of developmental patterns in sporangia and pollen grains (Endress, 2005), particularly since they can set limits between taxa, determine affinities, and help in the assessment of classification systems (Herr, 1984). Even though the phenotypic plasticity of embryological characters is small, the high species richness and the wide distribution of Bromeliaceae may affect the embryology of this group. Scarcity of embryological studies on androecium development in Bromeliaceae, particularly in Pitcairnioideae, taking into account also the restricted distribution of still one single population of D. pseudococcinea, prompted us to conduct the present investigation.
S.P. Mendes et al. / Flora 207 (2012) 622–627
Specifically, successful in vitro propagation experiments with the goal of conserving D. pseudococcinea will depend on data establishing the relationships between the size of flower and respective developmental stages of anthers based on anther wall development, androsporogenesis and androgametogenesis. Moreover, characterizing the embryological characters of D. pseudococcinea will advance our knowledge of Pitcairnioideae embryology and thus help to assess the relationship among taxa in the context of this subfamily. Material and methods Flowers and flower buds of D. pseudococcinea were collected at different developmental stages and fixed at room temperature in glutaraldehyde 2.5%, in sodium phosphate buffer 0.1 M pH 7.2, following Gabriel (1982). They were then dehydrated in an ethanol series and embedded in hydroxyethylmethacrylate, following Gerrits and Smid (1983). Transverse sections were cut to 2–3 m in thickness using a D-profile microtome knife in a Shandon Hypercut microtome, affixed to glass slides and stained with Toluidine Blue O 0.05% (O’Brien et al., 1965). Observations and images were made in an Olympus BX-50 optical microscope equipped with a CoolsnapPro digital camera. Furthermore, 0.5% Aniline Blue (Oparka and Read, 1994) and DAPI (Ruzin, 1999) were used to observe callose and the nuclei of generative and vegetative cells, respectively, under an Olympus BX-50 fluorescence microscope. For scanning electron microscopy (SEM), the anthers, after being fixed, were dehydrated in an ethanol series, mixed in the proportions of 3:1, 1:1 and 1:3 of ethanol and acetone, and washed twice in pure acetone. Then, they were critical-point dried using a 030 Leica EM critical point dryer, mounted onto pin stubs and coated with gold using a Emitech K550X sputter coater. Observations and electron micrographs were made with an EVO 40 SEM at 15 kV. Results The staminal primordia of D. pseudococcinea, in transverse section, are round-shaped with a meristematic structure differentiated into three layers: epidermal, subepidermal and central (Fig. 1). Thereafter, the anther assumes a bilobate shape (Fig. 2), whereas the epidermal layer follows the primordium growth through anticlinal divisions that characterize the epidermis. Cells of the subepidermal and central layers multiply by anticlinal, periclinal and oblique divisions. Next, the anther becomes tetralobate (Fig. 3), and the future sporangia become prominent. The cells of the subepidermal layer undergo periclinal divisions, originating two new layers: primary parietal and sporogenous, while the cells of the central layer originate the connective and vascular bundles. After the establishment of the primary parietal layer, its cells undergo further periclinal divisions, originating the outer and inner secondary parietal layers (Fig. 4). The cells of the outer secondary parietal layer divide periclinally, originating the outer secondary parietal layers 1 and 2. Parallel to those divisions, the cells of the inner secondary parietal layer also undergo periclinal divisions, originating the inner secondary parietal layers 1 and 2 (Fig. 5). Next, the anther wall exhibits five parietal layers: epidermis, originated from the epidermal layer; endothecium, originated from the outer secondary parietal layer 1; two middle layers, originated from outer secondary parietal layer 2 and inner secondary parietal layer 1; and tapetum, originated from the inner secondary parietal layer 2 (Fig. 6). However, in addition to the aforementioned origin of the tapetum, its inner part, which is adjacent to the connective tissue, originates from cells of the sporogenous tissue (subepidermal layer) (Fig. 5). These cells differ from the archesporial cells
623
Table 1 Relationships between the size of flower buds and flowers and the developmental stages of anthers in Dyckia pseudococcinea. Flower buds and flowers (mm)
Developmental stages
1–1.4 2–2.5
*Meristematic layers (l1, l2, l3) (Fig. 1) *CPP division in CPSI and CPSE; *archesporial initials (Fig. 4)
2.8–3.2
*Further divisions of parietal layers; *archesporial cells (Fig. 5) *Androspore mother cells (Fig. 6) *Dyads and tetrads (Figs. 7, 10–12) *Androspores (Fig. 13)
3.4–4 4.3–4.9 5–5.7 6.4–7.1 7.5–9.2 10.2–12
*Vacuolated androspores (Fig. 14) *Bicellular pollen grains (initial), without storage reserves (Fig. 15) *Mature pollen grains, with storage reserves; *anther dehiscence (Figs. 8, 16–18)
Parietal layers
Androsporogenesis
Androgametogenesis
by their smaller volume and rectangular, instead of isodiametric, shape (Fig. 5). Following the anther wall development, the secretory tapetum surrounds the sporogenous tissue, with some of its cells becoming binucleate (Fig. 6), starting their degeneration process at the androspore (microspore) dyad stage (Fig. 7). By the absence of further cellular divisions at advanced stages, the middle layers are compressed by the growth of the tapetum and sporogenous tissue and reabsorbed at the end of androsporogenesis (Fig. 8). Furthermore, the total degeneration of the tapetum is observed, whereas the endothecium exhibits particular parietal thickening of the helicoidal type (Fig. 9). Parallel to the development of the parietal layers, the differentiation of the archesporial initials (Fig. 4) is observed in the sporogenous layer, and at the end of their mitotic divisions, they mature into archesporial cells (Fig. 5). Those cells then undergo a meiotic prophase and are denominated androspore mother cells (Fig. 6) that begin to exhibit callose deposition. Next, the androspore mother cells undergo meiosis of the successive type, forming dyads (Figs. 7 and 10) after meiosis I and tetrads of isobilateral (Fig. 11) and decussate types (Fig. 12) after meiosis II. Then, callose dissolution liberates the androspores in the anther loculus, and the free androspores increase in volume (Fig. 13) and form vacuoles (Fig. 14) which push the nucleus toward the sporoderm. Next, the first asymmetric mitotic division of the androspore, now the androphyte mother cell, takes place, which, after cytokinesis, originates two cells with unequal volume: the vegetative and the generative cells (Fig. 15). The generative cell of reduced size is compressed by the vegetative cell against the sporoderm, which embodies it through a ‘strangulation’ process of its parietal surface (Fig. 15). After the generative cell is completely embodied by the vegetative cell, the vegetative nucleus and the generative cell, the latter exhibiting a fusiform aspect at this stage (Fig. 16), undergo a gradual approximation, characterizing the male germ unit (Fig. 17). This morphological unit persists until longitudinal anther dehiscence, which results in the dispersal of bicellular pollen grains (Fig. 18). The pollen grains are oblate, monosulcate (Figs. 19–20) and exhibit a microreticulate exine, which consists of lumina with variable outlines and shapes (Fig. 21). Thus, while the distal face of the pollen presents lumina with reduced dimensions (Fig. 19), the proximal face stands out by having lumina with comparatively larger dimensions (Fig. 20). The relationship between the sizes of flower buds and flowers and the developmental stages of the anthers are detailed in Table 1.
624
S.P. Mendes et al. / Flora 207 (2012) 622–627
Figs. 1–9. Anther wall formation of Dyckia pseudococcinea in transverse section. (1) Staminal primordia with meristematic layers: epidermal (l1), subepidermal (l2) and central (l3). (2) Bilobate anther. (3) Tetralobate anther; asterisks indicate sporangia. (4) Anther showing archesporial initials (AI) and the periclinal division of the primary parietal layer (PPL) into outer secondary parietal layer (OSPL) and inner secondary parietal layer (ISPL). (5) Sporangium with archesporial cells (AC); periclinal division of the OSPL into OSPL 1 and 2 (white arrow) and periclinal division of the ISPL into ISPL 1 and 2 (black arrow); asterisks indicate archesporial initials differentiating to form the tapetum. (6) Anther evidencing the androspore mother cells (AMC) and parietal layers: epidermis (EP), endothecium (END), middle layers (ML), tapetum (TP); binucleate cells of the tapetum are indicated by arrows. (7) Sporangium evidencing tapetum cells degenerating. (8) Mature tetralobate anther with specialized thickening of the endothecium (arrows). (9) Detail of the endothecium thickening of the helicoidal type in longitudinal section. Scale bar = 50 m (Figs. 6 and 9); 100 m (Figs. 1–5, 7 and 8).
Discussion Although studies reporting the development of anthers and pollen grains are scarce in Bromeliaceae (Lakshman, 1967; Rao and Wee, 1979; Sajo et al., 2005; Wee and Rao, 1974), this family can be generally characterized by anther wall development of the monocotyledonous type, secretory tapetum,
successive cytokinesis, linear or isobilateral tetrads (Johri et al., 1992), and monosulcate and bicellular pollen grains (Halbritter, 1992). The monocotyledonous type of anther development, considered a characteristic pattern among Bromeliaceae (Davis, 1966; Sajo et al., 2005), is a common feature in monocotyledons, and it is observed in most species of Poales (Johri et al., 1992).
S.P. Mendes et al. / Flora 207 (2012) 622–627
625
Figs. 10–18. Androsporogenesis and androgametogenesis of Dyckia pseudococcinea in transverse section. (10) Dyad of androspores. (11) Isobilateral tetrad of androspores. (12) Decussate tetrad of androspores evidencing the callose (fluorescence microscopy). (13) Detail of the free androspore. (14) Free androspore with vacuoles (arrows). (15) Embodiment of the generative cell (arrow) and nucleus of the vegetative cell (asterisk). (16) Bicellular pollen grain evidencing the fusiform generative cell (arrow) and the nucleus of the vegetative cell (asterisk). (17) Bicellular pollen grain, with spherical generative cell (arrow) and nucleus of the vegetative cell (asterisk). (18) Bicellular pollen grain, evidencing the nucleus of the vegetative cell (nvc) and the nucleus of the generative cell (ngc) (fluorescence microscopy). Scale bar = 50 m (Figs. 10–18).
However, the results obtained for D. pseudococcinea give evidence for a basic-type development, which was described as characteristic of basal families close to Bromeliaceae, such as Mayacaceae (Venturelli and Bouman, 1986) and Rapateaceae (Venturelli and Bouman, 1988). Therefore, if (i) basal families exhibit basictype development and (ii) most studied Bromeliaceae exhibit the monocotyledonous-type development, then the basic-type found in D. pseudococcinea suggests a reversion to the plesiomorphic state,
considering the derivative state of Dyckia Schult.f. species in the phylogeny of Bromeliaceae (Givnish et al., 2011). However, even with the predominance of monocotyledonous-type development among species of Bromeliaceae (Sajo et al., 2005), this character should not absolutely be generalized for the group. Hence, the need for more ontogenetic analyses in a higher number of Bromeliaceae species becomes evident, as an attempt to establish the relationship among taxa based on that characteristic.
626
S.P. Mendes et al. / Flora 207 (2012) 622–627
pseudococcinea, were interpreted as sterilization by Maheshwari (1950) or as somatization processes by Rutishauser (1969). Characteristics of sporogenous cells, such as their relative position within the sporangium, large size, dense cytoplasm, large nucleus and nucleolus (Schnarf, 1929), indicate a premeiotic cellular differentiation (PCD; Pozner, 2001). However, according to Pozner, PCD does not assure meiosis, but only indicates the possibility of future meiosis. According to this interpretation, the archesporial initials are only potentially able to follow the meiotic process and should therefore be interpreted as somatic cells until they reach the premeiotic G2 phase, when meiosis is considered irreversible (Ito and Takegami, 1982; Ninnemann and Epel, 1973). Hence, the archesporial initials keep their somatic lineage and may follow the path of cellular differentiation, meiotic or somatic, and the concepts of sterilization and somatization should not be used in this context. The specific developmental stage when there is embryogenic potential differs among species. In general, however, the period of sensitivity to inductive treatments is approximately the first pollen mitosis, i.e., between the vacuolated androspore stage and the initial bicellular pollen grain (Touraev et al., 2001), probably because the transcriptional status at that time is still proliferative and not yet completely differentiated (Malik et al., 2007). This relationship between the morphogenetic capacity of the tissues and their evolutionary stage has already been pointed out by Emons (1994), who suggested that the capacity of the explants to change their evolutionary pathway decreases with their stage of differentiation. Corroborating these interpretations, many studies suggest the vacuolated androspore stage as the most responsive to in vitro induction (Germanà et al., 2006; Peixe et al., 2004; Rajasekaran and Mullins, 1979; Solís et al., 2008). After the pollen grains begin to accumulate storage reserves, they usually lose their embryogenic capacity and follow the normal path of gametophytic development (Heberle-Bors, 1989; Raghavan, 1990). Based on this evidence, we suggest the preferential use of the vacuolated androspore stage, which is present in flower buds 6.4–7.1 mm in length, in protocols focused on the androgenesis of D. pseudococcinea. From the perspective of conserving this endangered plant species, the use of this stage in isolated androspores cultivated in vitro would make it possible to minimize the difficulties in obtaining new seedlings through in vitro propagation, thereby facilitating the reintroduction of D. pseudococcinea into the natural environment.
Figs. 19–21. SEM micrographs of pollen grain of Dyckia pseudococcinea. (19) Distal view showing the sulcus. (20) Proximal view. (21) Detailed view of the microreticulate exine. Scale bar = 10 m (Figs. 19–21).
The origin of the tapetum, as usually described in the literature, appears to be controversial. Interestingly, some reports suggest a dual origin, from the inner secondary parietal layer, and: (i) from the central layer of the stamen primordium, i.e., from the dedifferentiation of connective cells (Goldberg et al., 1993; Nanda and Gupta, 1978; Periasamy and Swamy, 1966) or (ii) from the ‘sterilization’ or ‘somatization’ of archesporial cells (Bhandari and Khosla, 1982; Boke, 1949). In D. pseudococcinea, both the outer and inner parts of the tapetum exhibit precursors derived from the subepidermal layer: the inner secondary parietal layer 2 and archesporial initials, respectively. Based on this evidence, it can be affirmed that the tapetum of D. pseudococcinea has a single origin, contrary to results reported in the literature. Hence, the statement of Gupta and Nanda (1978) that the double origin of the tapetum is a universal pattern should be reconsidered. Cases of cells with an origin that supposes a meiotic fate, but that differentiate as somatic cells, as observed in D.
Acknowledgements The authors thank the Programa de Pós-graduac¸ão em Botânica do Museu Nacional/UFRJ and the Instituto de Pesquisas Jardim Botânico do Rio de Janeiro (JBRJ). CAPES granted the first author a Master’s scholarship; CNPq granted the second author a fellowship. This study was funded by FAPERJ (process no. 112.349/2008).
References Bhandari, N.N., Khosla, R., 1982. Development and histochemistry of anther in Triticale cv. Tri-1. I. Some new aspects in early ontogeny. Phytomorphology 32, 18–27. Boke, N.H., 1949. Development of the stamens and carpels in Vinca rosea L. Am. J. Bot. 36, 535–547. Davis, G.L., 1966. Systematic Embryology of the Angiosperms. Wiley, New York. Emons, A.M.C., 1994. Somatic embryogenesis: cell biological aspects. Acta Bot. Neerl. 43, 1–14. Endress, P.K., 2005. Links between embryology and evolutionary floral morphology. Curr. Sci. 89, 749–754. Gabriel, B.L., 1982. Biological Electron Microscopy. Van Nostrand Reinhold, New York. ˜ M.C., 2006. Germanà, M.A., Chiancone, B., Levy-Guarda, N., Testillano, P.S., Risueno, Development of multicellular pollen of Eriobotrya japonica Lindl. through anther culture. Plant Sci. 171, 718–725.
S.P. Mendes et al. / Flora 207 (2012) 622–627 Gerrits, P.O., Smid, L., 1983. A new, less toxic polymerization system for the embedding of soft tissues in glycol methacrylate and subsequent preparing of serial sections. J. Microsc. 132, 81–85. Givnish, T.J., et al., 2011. Phylogeny, adaptive radiation, and historical biogeography in Bromeliaceae: insights from an eight-locus plastid phylogeny. Am. J. Bot. 98, 872–895. Goldberg, K.S., Beals, T.P., Sanders, P.M., 1993. Anther development: basic principles and practical applications. Plant Cell 5, 1217–1229. Gribaudo, I., Gambino, G., Vallania, R., 2004. Somatic embryogenesis from grapevine anthers: the optimal developmental stage for collecting explants. Am. J. Enol. Viticult. 55, 427–430. Gupta, S.C., Nanda, K., 1978. Ontogeny and histochemistry of dimorphic tapetum in Tecoma stans anthers. Bull. Soc. Bot. Fr. (1/2), 129–134. Halbritter, H., 1992. Morphology and systematic significance of Bromeliaceae pollen. Grana 31, 197–212. Heberle-Bors, E., 1989. Isolated pollen culture in tobacco: plant reproductive development in a nutshell. Sex. Plant Reprod. 2, 1–10. Herr, J.M., 1984. Embryology and taxonomy. In: Johri, B.M. (Ed.), Embryology of Angiosperms. Springer, Berlin, pp. 647–696. Hoffmann, F., Thomas, E., Wenzel, G., 1982. Anther culture as a breeding tool in rape. Theor. Appl. Genet. 61, 225–232. Ito, M., Takegami, M.H., 1982. Commitment of mitotic cells to meiosis during the G2 phase of premeiosis. Plant Cell Physiol. 23, 943–952. Johri, B.M., Ambegaokar, K.B., Srivastava, P.S., 1992. Comparative Embryology of Angiosperms. Springer, Berlin. Lakshman, K.K., 1967. Embryological studies in Bromeliaceae. I. Lindmania penduliflora (Ch Wright) Stapf. Proc. Indian Acad. Sci. B: Biol. Sci. 65, 49–55. Maheshwari, P., 1950. An Introduction to the Embryology of Angiosperms. McGrawHill, New York. Malik, M.R., Wang, F., Dirpaul, J.M., Zhou, N., Polowick, P.L., Ferrie, A.M.R., Krochko, J.E., 2007. Transcript profiling and identification of molecular markers for early microspore embryogenesis in Brassica napus. Plant Physiol. 144, 134–154. Mantovani, A., Iglesias, R.R., 2001. Terrestrial bromeliads of the “restinga” of “Barra de Maricá”, Rio de Janeiro: influence on the microclimate, soil and nutrient storage on the border of the vegetation islands. Leandra 16, 17–37. Martinelli, G., Vieira, C.M., Gonzáles, M., Leitman, P., Piratininga, A., Costa, A.F., Forzza, R., 2008. Bromeliaceae da Mata Atlântica Brasileira: lista de espécies, distribuic¸ão e conservac¸ão. Rodriguésia 59, 209–258. MMA – Ministério do Meio Ambiente, 2008. Lista Oficial das Espécies da Flora Brasileira Ameac¸adas de Extinc¸ão., http://www.mma.gov.br. Nanda, K., Gupta, S.C., 1978. Studies in the Bignoniaceae. II. Ontogeny of dimorphic anther tapetum in Tecoma. Am. J. Bot. 65, 400–405. Ninnemann, H., Epel, B., 1973. Inhibition of cell division by blue light. Exp. Cell Res. 79, 318–326. O’Brien, T.P., Feder, N., Mccully, M.E., 1965. Polychromatic staining of plant cell walls by Toluidine Blue O. Protoplasma 59, 368–373. Oparka, K.J., Read, N.D., 1994. The use of fluorescent probes for studies of living plant cells. In: Harris, N., Oparka, K.J. (Eds.), Plant Cell Biology – A Practical Approach. Oxford University Press, New York, pp. 27–50.
627
Peixe, A., Barroso, J., Potes, A., Pais, M.S., 2004. Induction of haploid morphogenic calluses from in vitro cultured anthers of Prunus armeniaca cv. ‘Harcot’. Plant Cell Tiss. Org. Cult. 77, 35–41. Periasamy, K., Swamy, B.G.L., 1966. Morphology of the anther tapetum in angiosperms. Curr. Sci. 35, 427–430. Pozner, R., 2001. Approach to the early sporangial development in angiosperms considering meiosis control and cellular differentiation. Plant Syst. Evol. 230, 25–42. Raghavan, V., 1990. From microspore to embryo: faces of the angiosperm pollen grain. In: Nijkamp, H.J.J., van der Plas, L.H., van Hartrigik, J. (Eds.), Progress in Plant Cellular and Molecular Biology. I.A.P.T.C., Kluwer, Dordrecht, pp. 213–221. Rajasekaran, K., Mullins, M.G., 1979. Embryos and plantlets from cultured anthers of hybrid grapevines. J. Exp. Bot. 30, 399–407. Rao, A.N., Wee, Y.C., 1979. Embryology of the pineapple, Ananas comosus (L.) Merr. New Phytol. 83, 485–497. Ribeiro, M.C., Metzger, J.P., Martensen, A.C., Ponzoni, F., Hirota, M.M., 2009. Brazilian Atlantic forest: how much is left and how is the remaining forest distributed? Implications for conservation. Biol. Conserv. 142, 1141–1153. Rutishauser, A., 1969. Embryologie und Fortpflanzungsbiologie der Angiospermen. Eine Einführung. Springer, Wien. Ruzin, S.E., 1999. Plant Microtechnique and Microscopy. Oxford University Press, New York. Sajo, M.G., Furness, C.A., Prychid, C.J., Rudall, P.J., 2005. Microsporogenesis and anther development in Bromeliaceae. Grana 44, 65–74. Scarano, F.R., 2009. Plant communities at the periphery of the Atlantic rain forest: rare-species bias and its risks for conservation. Biol. Conserv. 142, 1201–1208. Schnarf, K., 1929. Embryologie der Angiospermen. Handbuch der Pflanzenanatomie, 2 Abt., 2 Teil. Borntraeger, Berlin. Siebel, J., Pauls, K.P., 1989. A comparison of anther and microspore culture as a breeding tool in Brassica napus. Theor. Appl. Genet. 78, 473–479. ˜ M.C., Testillano, P.S., Solís, M.T., Pintos, B., Prado, M.J., Bueno, M.A., Raska, I., Risueno, 2008. Early markers of in vitro microspore reprogramming to embryogenesis in olive (Olea europaea L.). Plant Sci. 174, 597–605. Touraev, A., Pfosser, M., Heberle-Bors, E., 2001. The microspore: a haploid multipurpose cell. Adv. Bot. Res. 35, 53–109. Venturelli, M., Bouman, F., 1986. Embryology and seed development in Mayaca fluviatilis (Mayacaceae). Acta Bot. Neerl. 35, 497–516. Venturelli, M., Bouman, F., 1988. Development of ovule and seed in Rapateaceae. Bot. J. Linn. Soc. 97, 267–294. ˜ M., Segura, A., González-Benito, Vidal, J.R., Rama, J., Taboada, L., Martin, C., Ibanez, M.E., 2009. Improved somatic embryogenesis of grapevine (Vitis vinifera) with focus on induction parameters and efficient plant regeneration. Plant Cell Tiss. Org. Cult. 96, 85–94. Wee, Y.C., Rao, A.N., 1974. Gametophytes and seed development in pineapple. Curr. Sci. 43, 171–173.
Contents lists available at SciVerse ScienceDirect
Flora journal homepage: www.elsevier.de/flora
Androecium development in the bromeliad Dyckia pseudococcinea L.B.Sm. (Pitcairnioideae-Bromeliaceae), an endangered species endemic to Brazil: implications for conservation Simone Petrucci Mendes a,∗ , Cecília G. Costa b , Karen L.G. De Toni b a b
Museu Nacional/Universidade Federal do Rio de Janeiro, Quinta da Boa Vista s.n., São Cristovão, Rio de Janeiro, RJ, Brazil Instituto de Pesquisas Jardim Botânico do Rio de Janeiro, Diretoria de Pesquisa, Pacheco Leão 915, Jardim Botânico, Rio de Janeiro, RJ, Brazil
a r t i c l e
i n f o
Article history: Received 9 December 2011 Received in revised form 5 May 2012 Accepted 26 May 2012 Keywords: Anther Tapetum Androsporogenesis Androgametogenesis Pollen grains Conservation
a b s t r a c t Dyckia pseudococcinea L.B.Sm. is endemic to the restingas of Maricá, State of Rio de Janeiro, in southeastern Brazil. However, since this area is under intense ecological stress, D. pseudococcinea is considered an endangered plant species. An important step toward the conservation of this species is establishing the developmental stage most favorable to in vitro propagation. Accordingly, the present study analyzed the androecium to establish the developmental stages of anthers, emphasizing anther wall development, androsporogenesis and androgametogenesis. The relationships between the size of flowers and anthers were also studied. Anther wall development follows the basic-type, while the tapetum, which is originated from the subepidermal layer (inner secondary parietal layer 2 and archesporial initials), follows the secretory-type. Androsporogenesis is successive and originates isobilateral and decussate tetrads. The oblate and monosulcate pollen grains exhibit microreticulate exine and are dispersed at the bicellular stage. At the vacuolated stage, the androspore (microspore) is still proliferating and not yet completely differentiated. Therefore, since the androspore has not yet lost its embryonic capacity, we suggest the preferential use of this developmental stage, which is present in flower buds 6.4–7.1 mm in length, in conservation protocols focused on the androgenesis of D. pseudococcinea. © 2012 Elsevier GmbH. All rights reserved.
Introduction Bromeliaceae is one of the most common families in restinga ecosystems of the Brazilian Atlantic Forest (Mantovani and Iglesias, 2001), with more than one third of the species being endemic to this biome (Martinelli et al., 2008). However, only 12% of the forest original area remains, mainly because of uncontrolled heavy exploration for commercial purposes and the associated environmental degradation (Ribeiro et al., 2009). This has strongly contributed to the dramatic reduction, or even extinction, of many species, also for the Bromeliaceae. Therefore, rare and endemic species are now frequently targets of conservation initiatives, since they are particularly vulnerable and prone to the risk of extinction (Scarano, 2009), in some cases being also keystone species or flagship taxa that sensitize the public for the ecological deterioration. Dyckia pseudococcinea L.B.Sm. belongs to such taxa and is included in the Brazilian List of Threatened Plant Species (MMA, 2008), mainly because of
∗ Corresponding author at: Instituto de Pesquisas Jardim Botânico do Rio de Janeiro, Diretoria de Pesquisa, Rua Pacheco Leão 915/sala 306, CEP 22460-030, Jardim Botânico, Rio de Janeiro, RJ, Brazil. Tel.: +55 21 3204 2099. E-mail address: [email protected] (S.P. Mendes). 0367-2530/$ – see front matter © 2012 Elsevier GmbH. All rights reserved. http://dx.doi.org/10.1016/j.flora.2012.06.016
its endemism to the restingas of Maricá, State of Rio de Janeiro, in southeastern Brazil, an area under intense ecological stress. Aimed at species conservation, in vitro culture of anthers has been broadly used in the production of haploid and double haploid plants for reintroduction in degraded areas (Hoffmann et al., 1982; Siebel and Pauls, 1989). It is considered a means to avoid ultimate extinction of plant species, albeit at a low level of genetic diversity. However, correctly identifying the stages of anther development is critical to the success of in vitro cultures (Gribaudo et al., 2004; Vidal et al., 2009). As such, understanding the ontogenetic details of anther development is a precondition in propagation studies. At the same time, however, evolutionary analysis in angiosperms depends generally upon an understanding of developmental patterns in sporangia and pollen grains (Endress, 2005), particularly since they can set limits between taxa, determine affinities, and help in the assessment of classification systems (Herr, 1984). Even though the phenotypic plasticity of embryological characters is small, the high species richness and the wide distribution of Bromeliaceae may affect the embryology of this group. Scarcity of embryological studies on androecium development in Bromeliaceae, particularly in Pitcairnioideae, taking into account also the restricted distribution of still one single population of D. pseudococcinea, prompted us to conduct the present investigation.
S.P. Mendes et al. / Flora 207 (2012) 622–627
Specifically, successful in vitro propagation experiments with the goal of conserving D. pseudococcinea will depend on data establishing the relationships between the size of flower and respective developmental stages of anthers based on anther wall development, androsporogenesis and androgametogenesis. Moreover, characterizing the embryological characters of D. pseudococcinea will advance our knowledge of Pitcairnioideae embryology and thus help to assess the relationship among taxa in the context of this subfamily. Material and methods Flowers and flower buds of D. pseudococcinea were collected at different developmental stages and fixed at room temperature in glutaraldehyde 2.5%, in sodium phosphate buffer 0.1 M pH 7.2, following Gabriel (1982). They were then dehydrated in an ethanol series and embedded in hydroxyethylmethacrylate, following Gerrits and Smid (1983). Transverse sections were cut to 2–3 m in thickness using a D-profile microtome knife in a Shandon Hypercut microtome, affixed to glass slides and stained with Toluidine Blue O 0.05% (O’Brien et al., 1965). Observations and images were made in an Olympus BX-50 optical microscope equipped with a CoolsnapPro digital camera. Furthermore, 0.5% Aniline Blue (Oparka and Read, 1994) and DAPI (Ruzin, 1999) were used to observe callose and the nuclei of generative and vegetative cells, respectively, under an Olympus BX-50 fluorescence microscope. For scanning electron microscopy (SEM), the anthers, after being fixed, were dehydrated in an ethanol series, mixed in the proportions of 3:1, 1:1 and 1:3 of ethanol and acetone, and washed twice in pure acetone. Then, they were critical-point dried using a 030 Leica EM critical point dryer, mounted onto pin stubs and coated with gold using a Emitech K550X sputter coater. Observations and electron micrographs were made with an EVO 40 SEM at 15 kV. Results The staminal primordia of D. pseudococcinea, in transverse section, are round-shaped with a meristematic structure differentiated into three layers: epidermal, subepidermal and central (Fig. 1). Thereafter, the anther assumes a bilobate shape (Fig. 2), whereas the epidermal layer follows the primordium growth through anticlinal divisions that characterize the epidermis. Cells of the subepidermal and central layers multiply by anticlinal, periclinal and oblique divisions. Next, the anther becomes tetralobate (Fig. 3), and the future sporangia become prominent. The cells of the subepidermal layer undergo periclinal divisions, originating two new layers: primary parietal and sporogenous, while the cells of the central layer originate the connective and vascular bundles. After the establishment of the primary parietal layer, its cells undergo further periclinal divisions, originating the outer and inner secondary parietal layers (Fig. 4). The cells of the outer secondary parietal layer divide periclinally, originating the outer secondary parietal layers 1 and 2. Parallel to those divisions, the cells of the inner secondary parietal layer also undergo periclinal divisions, originating the inner secondary parietal layers 1 and 2 (Fig. 5). Next, the anther wall exhibits five parietal layers: epidermis, originated from the epidermal layer; endothecium, originated from the outer secondary parietal layer 1; two middle layers, originated from outer secondary parietal layer 2 and inner secondary parietal layer 1; and tapetum, originated from the inner secondary parietal layer 2 (Fig. 6). However, in addition to the aforementioned origin of the tapetum, its inner part, which is adjacent to the connective tissue, originates from cells of the sporogenous tissue (subepidermal layer) (Fig. 5). These cells differ from the archesporial cells
623
Table 1 Relationships between the size of flower buds and flowers and the developmental stages of anthers in Dyckia pseudococcinea. Flower buds and flowers (mm)
Developmental stages
1–1.4 2–2.5
*Meristematic layers (l1, l2, l3) (Fig. 1) *CPP division in CPSI and CPSE; *archesporial initials (Fig. 4)
2.8–3.2
*Further divisions of parietal layers; *archesporial cells (Fig. 5) *Androspore mother cells (Fig. 6) *Dyads and tetrads (Figs. 7, 10–12) *Androspores (Fig. 13)
3.4–4 4.3–4.9 5–5.7 6.4–7.1 7.5–9.2 10.2–12
*Vacuolated androspores (Fig. 14) *Bicellular pollen grains (initial), without storage reserves (Fig. 15) *Mature pollen grains, with storage reserves; *anther dehiscence (Figs. 8, 16–18)
Parietal layers
Androsporogenesis
Androgametogenesis
by their smaller volume and rectangular, instead of isodiametric, shape (Fig. 5). Following the anther wall development, the secretory tapetum surrounds the sporogenous tissue, with some of its cells becoming binucleate (Fig. 6), starting their degeneration process at the androspore (microspore) dyad stage (Fig. 7). By the absence of further cellular divisions at advanced stages, the middle layers are compressed by the growth of the tapetum and sporogenous tissue and reabsorbed at the end of androsporogenesis (Fig. 8). Furthermore, the total degeneration of the tapetum is observed, whereas the endothecium exhibits particular parietal thickening of the helicoidal type (Fig. 9). Parallel to the development of the parietal layers, the differentiation of the archesporial initials (Fig. 4) is observed in the sporogenous layer, and at the end of their mitotic divisions, they mature into archesporial cells (Fig. 5). Those cells then undergo a meiotic prophase and are denominated androspore mother cells (Fig. 6) that begin to exhibit callose deposition. Next, the androspore mother cells undergo meiosis of the successive type, forming dyads (Figs. 7 and 10) after meiosis I and tetrads of isobilateral (Fig. 11) and decussate types (Fig. 12) after meiosis II. Then, callose dissolution liberates the androspores in the anther loculus, and the free androspores increase in volume (Fig. 13) and form vacuoles (Fig. 14) which push the nucleus toward the sporoderm. Next, the first asymmetric mitotic division of the androspore, now the androphyte mother cell, takes place, which, after cytokinesis, originates two cells with unequal volume: the vegetative and the generative cells (Fig. 15). The generative cell of reduced size is compressed by the vegetative cell against the sporoderm, which embodies it through a ‘strangulation’ process of its parietal surface (Fig. 15). After the generative cell is completely embodied by the vegetative cell, the vegetative nucleus and the generative cell, the latter exhibiting a fusiform aspect at this stage (Fig. 16), undergo a gradual approximation, characterizing the male germ unit (Fig. 17). This morphological unit persists until longitudinal anther dehiscence, which results in the dispersal of bicellular pollen grains (Fig. 18). The pollen grains are oblate, monosulcate (Figs. 19–20) and exhibit a microreticulate exine, which consists of lumina with variable outlines and shapes (Fig. 21). Thus, while the distal face of the pollen presents lumina with reduced dimensions (Fig. 19), the proximal face stands out by having lumina with comparatively larger dimensions (Fig. 20). The relationship between the sizes of flower buds and flowers and the developmental stages of the anthers are detailed in Table 1.
624
S.P. Mendes et al. / Flora 207 (2012) 622–627
Figs. 1–9. Anther wall formation of Dyckia pseudococcinea in transverse section. (1) Staminal primordia with meristematic layers: epidermal (l1), subepidermal (l2) and central (l3). (2) Bilobate anther. (3) Tetralobate anther; asterisks indicate sporangia. (4) Anther showing archesporial initials (AI) and the periclinal division of the primary parietal layer (PPL) into outer secondary parietal layer (OSPL) and inner secondary parietal layer (ISPL). (5) Sporangium with archesporial cells (AC); periclinal division of the OSPL into OSPL 1 and 2 (white arrow) and periclinal division of the ISPL into ISPL 1 and 2 (black arrow); asterisks indicate archesporial initials differentiating to form the tapetum. (6) Anther evidencing the androspore mother cells (AMC) and parietal layers: epidermis (EP), endothecium (END), middle layers (ML), tapetum (TP); binucleate cells of the tapetum are indicated by arrows. (7) Sporangium evidencing tapetum cells degenerating. (8) Mature tetralobate anther with specialized thickening of the endothecium (arrows). (9) Detail of the endothecium thickening of the helicoidal type in longitudinal section. Scale bar = 50 m (Figs. 6 and 9); 100 m (Figs. 1–5, 7 and 8).
Discussion Although studies reporting the development of anthers and pollen grains are scarce in Bromeliaceae (Lakshman, 1967; Rao and Wee, 1979; Sajo et al., 2005; Wee and Rao, 1974), this family can be generally characterized by anther wall development of the monocotyledonous type, secretory tapetum,
successive cytokinesis, linear or isobilateral tetrads (Johri et al., 1992), and monosulcate and bicellular pollen grains (Halbritter, 1992). The monocotyledonous type of anther development, considered a characteristic pattern among Bromeliaceae (Davis, 1966; Sajo et al., 2005), is a common feature in monocotyledons, and it is observed in most species of Poales (Johri et al., 1992).
S.P. Mendes et al. / Flora 207 (2012) 622–627
625
Figs. 10–18. Androsporogenesis and androgametogenesis of Dyckia pseudococcinea in transverse section. (10) Dyad of androspores. (11) Isobilateral tetrad of androspores. (12) Decussate tetrad of androspores evidencing the callose (fluorescence microscopy). (13) Detail of the free androspore. (14) Free androspore with vacuoles (arrows). (15) Embodiment of the generative cell (arrow) and nucleus of the vegetative cell (asterisk). (16) Bicellular pollen grain evidencing the fusiform generative cell (arrow) and the nucleus of the vegetative cell (asterisk). (17) Bicellular pollen grain, with spherical generative cell (arrow) and nucleus of the vegetative cell (asterisk). (18) Bicellular pollen grain, evidencing the nucleus of the vegetative cell (nvc) and the nucleus of the generative cell (ngc) (fluorescence microscopy). Scale bar = 50 m (Figs. 10–18).
However, the results obtained for D. pseudococcinea give evidence for a basic-type development, which was described as characteristic of basal families close to Bromeliaceae, such as Mayacaceae (Venturelli and Bouman, 1986) and Rapateaceae (Venturelli and Bouman, 1988). Therefore, if (i) basal families exhibit basictype development and (ii) most studied Bromeliaceae exhibit the monocotyledonous-type development, then the basic-type found in D. pseudococcinea suggests a reversion to the plesiomorphic state,
considering the derivative state of Dyckia Schult.f. species in the phylogeny of Bromeliaceae (Givnish et al., 2011). However, even with the predominance of monocotyledonous-type development among species of Bromeliaceae (Sajo et al., 2005), this character should not absolutely be generalized for the group. Hence, the need for more ontogenetic analyses in a higher number of Bromeliaceae species becomes evident, as an attempt to establish the relationship among taxa based on that characteristic.
626
S.P. Mendes et al. / Flora 207 (2012) 622–627
pseudococcinea, were interpreted as sterilization by Maheshwari (1950) or as somatization processes by Rutishauser (1969). Characteristics of sporogenous cells, such as their relative position within the sporangium, large size, dense cytoplasm, large nucleus and nucleolus (Schnarf, 1929), indicate a premeiotic cellular differentiation (PCD; Pozner, 2001). However, according to Pozner, PCD does not assure meiosis, but only indicates the possibility of future meiosis. According to this interpretation, the archesporial initials are only potentially able to follow the meiotic process and should therefore be interpreted as somatic cells until they reach the premeiotic G2 phase, when meiosis is considered irreversible (Ito and Takegami, 1982; Ninnemann and Epel, 1973). Hence, the archesporial initials keep their somatic lineage and may follow the path of cellular differentiation, meiotic or somatic, and the concepts of sterilization and somatization should not be used in this context. The specific developmental stage when there is embryogenic potential differs among species. In general, however, the period of sensitivity to inductive treatments is approximately the first pollen mitosis, i.e., between the vacuolated androspore stage and the initial bicellular pollen grain (Touraev et al., 2001), probably because the transcriptional status at that time is still proliferative and not yet completely differentiated (Malik et al., 2007). This relationship between the morphogenetic capacity of the tissues and their evolutionary stage has already been pointed out by Emons (1994), who suggested that the capacity of the explants to change their evolutionary pathway decreases with their stage of differentiation. Corroborating these interpretations, many studies suggest the vacuolated androspore stage as the most responsive to in vitro induction (Germanà et al., 2006; Peixe et al., 2004; Rajasekaran and Mullins, 1979; Solís et al., 2008). After the pollen grains begin to accumulate storage reserves, they usually lose their embryogenic capacity and follow the normal path of gametophytic development (Heberle-Bors, 1989; Raghavan, 1990). Based on this evidence, we suggest the preferential use of the vacuolated androspore stage, which is present in flower buds 6.4–7.1 mm in length, in protocols focused on the androgenesis of D. pseudococcinea. From the perspective of conserving this endangered plant species, the use of this stage in isolated androspores cultivated in vitro would make it possible to minimize the difficulties in obtaining new seedlings through in vitro propagation, thereby facilitating the reintroduction of D. pseudococcinea into the natural environment.
Figs. 19–21. SEM micrographs of pollen grain of Dyckia pseudococcinea. (19) Distal view showing the sulcus. (20) Proximal view. (21) Detailed view of the microreticulate exine. Scale bar = 10 m (Figs. 19–21).
The origin of the tapetum, as usually described in the literature, appears to be controversial. Interestingly, some reports suggest a dual origin, from the inner secondary parietal layer, and: (i) from the central layer of the stamen primordium, i.e., from the dedifferentiation of connective cells (Goldberg et al., 1993; Nanda and Gupta, 1978; Periasamy and Swamy, 1966) or (ii) from the ‘sterilization’ or ‘somatization’ of archesporial cells (Bhandari and Khosla, 1982; Boke, 1949). In D. pseudococcinea, both the outer and inner parts of the tapetum exhibit precursors derived from the subepidermal layer: the inner secondary parietal layer 2 and archesporial initials, respectively. Based on this evidence, it can be affirmed that the tapetum of D. pseudococcinea has a single origin, contrary to results reported in the literature. Hence, the statement of Gupta and Nanda (1978) that the double origin of the tapetum is a universal pattern should be reconsidered. Cases of cells with an origin that supposes a meiotic fate, but that differentiate as somatic cells, as observed in D.
Acknowledgements The authors thank the Programa de Pós-graduac¸ão em Botânica do Museu Nacional/UFRJ and the Instituto de Pesquisas Jardim Botânico do Rio de Janeiro (JBRJ). CAPES granted the first author a Master’s scholarship; CNPq granted the second author a fellowship. This study was funded by FAPERJ (process no. 112.349/2008).
References Bhandari, N.N., Khosla, R., 1982. Development and histochemistry of anther in Triticale cv. Tri-1. I. Some new aspects in early ontogeny. Phytomorphology 32, 18–27. Boke, N.H., 1949. Development of the stamens and carpels in Vinca rosea L. Am. J. Bot. 36, 535–547. Davis, G.L., 1966. Systematic Embryology of the Angiosperms. Wiley, New York. Emons, A.M.C., 1994. Somatic embryogenesis: cell biological aspects. Acta Bot. Neerl. 43, 1–14. Endress, P.K., 2005. Links between embryology and evolutionary floral morphology. Curr. Sci. 89, 749–754. Gabriel, B.L., 1982. Biological Electron Microscopy. Van Nostrand Reinhold, New York. ˜ M.C., 2006. Germanà, M.A., Chiancone, B., Levy-Guarda, N., Testillano, P.S., Risueno, Development of multicellular pollen of Eriobotrya japonica Lindl. through anther culture. Plant Sci. 171, 718–725.
S.P. Mendes et al. / Flora 207 (2012) 622–627 Gerrits, P.O., Smid, L., 1983. A new, less toxic polymerization system for the embedding of soft tissues in glycol methacrylate and subsequent preparing of serial sections. J. Microsc. 132, 81–85. Givnish, T.J., et al., 2011. Phylogeny, adaptive radiation, and historical biogeography in Bromeliaceae: insights from an eight-locus plastid phylogeny. Am. J. Bot. 98, 872–895. Goldberg, K.S., Beals, T.P., Sanders, P.M., 1993. Anther development: basic principles and practical applications. Plant Cell 5, 1217–1229. Gribaudo, I., Gambino, G., Vallania, R., 2004. Somatic embryogenesis from grapevine anthers: the optimal developmental stage for collecting explants. Am. J. Enol. Viticult. 55, 427–430. Gupta, S.C., Nanda, K., 1978. Ontogeny and histochemistry of dimorphic tapetum in Tecoma stans anthers. Bull. Soc. Bot. Fr. (1/2), 129–134. Halbritter, H., 1992. Morphology and systematic significance of Bromeliaceae pollen. Grana 31, 197–212. Heberle-Bors, E., 1989. Isolated pollen culture in tobacco: plant reproductive development in a nutshell. Sex. Plant Reprod. 2, 1–10. Herr, J.M., 1984. Embryology and taxonomy. In: Johri, B.M. (Ed.), Embryology of Angiosperms. Springer, Berlin, pp. 647–696. Hoffmann, F., Thomas, E., Wenzel, G., 1982. Anther culture as a breeding tool in rape. Theor. Appl. Genet. 61, 225–232. Ito, M., Takegami, M.H., 1982. Commitment of mitotic cells to meiosis during the G2 phase of premeiosis. Plant Cell Physiol. 23, 943–952. Johri, B.M., Ambegaokar, K.B., Srivastava, P.S., 1992. Comparative Embryology of Angiosperms. Springer, Berlin. Lakshman, K.K., 1967. Embryological studies in Bromeliaceae. I. Lindmania penduliflora (Ch Wright) Stapf. Proc. Indian Acad. Sci. B: Biol. Sci. 65, 49–55. Maheshwari, P., 1950. An Introduction to the Embryology of Angiosperms. McGrawHill, New York. Malik, M.R., Wang, F., Dirpaul, J.M., Zhou, N., Polowick, P.L., Ferrie, A.M.R., Krochko, J.E., 2007. Transcript profiling and identification of molecular markers for early microspore embryogenesis in Brassica napus. Plant Physiol. 144, 134–154. Mantovani, A., Iglesias, R.R., 2001. Terrestrial bromeliads of the “restinga” of “Barra de Maricá”, Rio de Janeiro: influence on the microclimate, soil and nutrient storage on the border of the vegetation islands. Leandra 16, 17–37. Martinelli, G., Vieira, C.M., Gonzáles, M., Leitman, P., Piratininga, A., Costa, A.F., Forzza, R., 2008. Bromeliaceae da Mata Atlântica Brasileira: lista de espécies, distribuic¸ão e conservac¸ão. Rodriguésia 59, 209–258. MMA – Ministério do Meio Ambiente, 2008. Lista Oficial das Espécies da Flora Brasileira Ameac¸adas de Extinc¸ão., http://www.mma.gov.br. Nanda, K., Gupta, S.C., 1978. Studies in the Bignoniaceae. II. Ontogeny of dimorphic anther tapetum in Tecoma. Am. J. Bot. 65, 400–405. Ninnemann, H., Epel, B., 1973. Inhibition of cell division by blue light. Exp. Cell Res. 79, 318–326. O’Brien, T.P., Feder, N., Mccully, M.E., 1965. Polychromatic staining of plant cell walls by Toluidine Blue O. Protoplasma 59, 368–373. Oparka, K.J., Read, N.D., 1994. The use of fluorescent probes for studies of living plant cells. In: Harris, N., Oparka, K.J. (Eds.), Plant Cell Biology – A Practical Approach. Oxford University Press, New York, pp. 27–50.
627
Peixe, A., Barroso, J., Potes, A., Pais, M.S., 2004. Induction of haploid morphogenic calluses from in vitro cultured anthers of Prunus armeniaca cv. ‘Harcot’. Plant Cell Tiss. Org. Cult. 77, 35–41. Periasamy, K., Swamy, B.G.L., 1966. Morphology of the anther tapetum in angiosperms. Curr. Sci. 35, 427–430. Pozner, R., 2001. Approach to the early sporangial development in angiosperms considering meiosis control and cellular differentiation. Plant Syst. Evol. 230, 25–42. Raghavan, V., 1990. From microspore to embryo: faces of the angiosperm pollen grain. In: Nijkamp, H.J.J., van der Plas, L.H., van Hartrigik, J. (Eds.), Progress in Plant Cellular and Molecular Biology. I.A.P.T.C., Kluwer, Dordrecht, pp. 213–221. Rajasekaran, K., Mullins, M.G., 1979. Embryos and plantlets from cultured anthers of hybrid grapevines. J. Exp. Bot. 30, 399–407. Rao, A.N., Wee, Y.C., 1979. Embryology of the pineapple, Ananas comosus (L.) Merr. New Phytol. 83, 485–497. Ribeiro, M.C., Metzger, J.P., Martensen, A.C., Ponzoni, F., Hirota, M.M., 2009. Brazilian Atlantic forest: how much is left and how is the remaining forest distributed? Implications for conservation. Biol. Conserv. 142, 1141–1153. Rutishauser, A., 1969. Embryologie und Fortpflanzungsbiologie der Angiospermen. Eine Einführung. Springer, Wien. Ruzin, S.E., 1999. Plant Microtechnique and Microscopy. Oxford University Press, New York. Sajo, M.G., Furness, C.A., Prychid, C.J., Rudall, P.J., 2005. Microsporogenesis and anther development in Bromeliaceae. Grana 44, 65–74. Scarano, F.R., 2009. Plant communities at the periphery of the Atlantic rain forest: rare-species bias and its risks for conservation. Biol. Conserv. 142, 1201–1208. Schnarf, K., 1929. Embryologie der Angiospermen. Handbuch der Pflanzenanatomie, 2 Abt., 2 Teil. Borntraeger, Berlin. Siebel, J., Pauls, K.P., 1989. A comparison of anther and microspore culture as a breeding tool in Brassica napus. Theor. Appl. Genet. 78, 473–479. ˜ M.C., Testillano, P.S., Solís, M.T., Pintos, B., Prado, M.J., Bueno, M.A., Raska, I., Risueno, 2008. Early markers of in vitro microspore reprogramming to embryogenesis in olive (Olea europaea L.). Plant Sci. 174, 597–605. Touraev, A., Pfosser, M., Heberle-Bors, E., 2001. The microspore: a haploid multipurpose cell. Adv. Bot. Res. 35, 53–109. Venturelli, M., Bouman, F., 1986. Embryology and seed development in Mayaca fluviatilis (Mayacaceae). Acta Bot. Neerl. 35, 497–516. Venturelli, M., Bouman, F., 1988. Development of ovule and seed in Rapateaceae. Bot. J. Linn. Soc. 97, 267–294. ˜ M., Segura, A., González-Benito, Vidal, J.R., Rama, J., Taboada, L., Martin, C., Ibanez, M.E., 2009. Improved somatic embryogenesis of grapevine (Vitis vinifera) with focus on induction parameters and efficient plant regeneration. Plant Cell Tiss. Org. Cult. 96, 85–94. Wee, Y.C., Rao, A.N., 1974. Gametophytes and seed development in pineapple. Curr. Sci. 43, 171–173.