Flower Morphologic Anatomy and Embryological Characteristics in Chrysanthemum Multicaule (Asteraceae)

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Flower morphologic anatomy and embryological characteristics in Chrysanthemum multicaule (Asteraceae)

ARTICLE in SCIENTIA HORTICULTURAE · MAY 2010 Impact Factor: 1.5 · DOI: 10.1016/j.scienta.2010.02.009

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Flower morphologic anatomy and embryological characteristics in Chrysanthemum multicaule (Asteraceae)

Yanming Deng, Sumei Chen, Nianjun Teng, Fadi Chen ∗, Fengtong Li, Aiping Song, Zhiyong Guan

College of Horticulture, Nanjing Agricultural University, Nanjing, Jiangsu 210095, China article info abstract

Article history: Chrysanthemum multicaule is an annual herbaceous ornamental species. The inflorescence is gynomo- Received 12 October 2009 noecious and consists of bisexual tubular florets and female ray florets. The pistils consist of two stigmas Received in revised form 8 February 2010 which are of the open type with a hollow stylar canal. At the base of the tubular floret style, the pistil is Accepted 9 February 2010 surrounded by oil gland cells. The anthers are tetrasporangiate and the young anther wall is composed of epidermis, endothecium, middle layer and tapetum. The mature anther wall comprises only thickened Keywords: endothecium after the release of the pollens. In the tubular florets, simultaneous microsporocyte meiotic Chrysanthemum multicaule cytokinesis results in mostly tetrahedral with a small proportion of decussate tetrads. The mature pollen Gametogenesis Sporogenesis grain is tricellular. The ovules are unitegmic and tenuinucellate, and the nucellus degenerates during the Embryogenesis development of the megasporocyte. The development of the embryo sac follows the Polygonum type. At Anatomy 4–6 days after blooming, the embryos reached the globular stage, thereafter passing through the heart- and torpedo-shape stages before maturing into the cotyledon embryos. From blooming to seed maturity, it takes about 3–4 weeks under our conditions. © 2010 Elsevier B.V. All rights reserved.

1. Introduction 2007). Here, we describe the morphology and anatomical structure of the florets of C. multicaule, and its sporogenesis, gametogenesis, Many Chrysanthemum species (Asteraceae-Anthemideae) are and embryogenesis, with a view to providing a firm basis for the exploited by the horticultural industry (Bremer and Humphries, utilization of this species. 1993). Chrysanthemum coronarium is a famous ornamental plant, while C. segetum is consumed as a common vegetable (Castellanos et al., 2001). The annual herbaceous species C. multicaule Desf. orig- 2. Materials and methods inates from Africa, and has been introduced to many countries. In China, it has become a particularly popular ornamental plant. The C. multicaule plants were grown at the Chrysanthemum species is highly fertile, flowers from mid-spring to early summer, Germplasm Resource Preserving Centre, Nanjing Agricultural Uni- grows to a height of 10–20 cm, and forms a dense mass of short versity, China. Inflorescences at various stages of development branches and leaves. As a result, it is an ideal subject for ground- were fixed in 5:5:90 formalin:acetic acid:70% ethanol (FAA), dehy- cover in borders, for filling flower beds and as a pot plant (Chen et drated through an alcohol series, infiltrated with xylene, and al., 1995; Zhao et al., 2009). The species has been shown to be highly embedded in paraffin wax as described by Li et al. (2009). Sections resistant to a spectrum of diseases and pests (Zhou et al., 2006). C. were cut to a thickness of 6–10 ␮m, stained in Heidenhain’s haema- multicaule is therefore regarded not only as a useful ornamental toxylin, and observed by optical microscope. The florets external plant in itself, but also as a potential reservoir of useful genetic morphology was observed directly under stereoscopic microscope. variation for chrysanthemum improvement via wide hybridization (Zhao et al., 2009). As far as we are aware, no systematic study has been made as 3. Results yet of either the reproductive organs of C. multicaule, or its embryological characteristics, even though this information is relevant 3.1. The morphology and anatomical structure of the flower to the understanding of angiosperm ontogenesis (Hu, 2005; Ao, The inflorescence is gynomonoecious, bearing both unisexual (female) ray florets and bisexual (hermaphroditic) tubular florets (Fig. 1A). The corolla of the ray floret consists of a single petal ∗ Corresponding author. Tel.: +86 25 84395231; fax: +86 25 84395266. (Fig. 1B), while the tubular bisexual florets are gamopetalous with E-mail address: [email protected] (F. Chen). five partially fused petals (Fig. 1C).

Fig. 1. Flowers of C. multicaule. ov, ovary; pe, petal; st, stigma. (A) A flower. (B) A ray floret. (C) A tubular floret. Scale bars: A = 10 mm, B=5mm,C=1mm.

Fig. 2. The anatomical structures of tubular and ray florets in C. multicaule. ova, ovary; ovu, ovule; pe, petal; sta, stamen; stb, style branch; sty, style. (A) The ray floret is composed of a single petal and two style branches. The arrow shows the hollow stylar canal. (B) The tubular floret has five stamens and five petals. The arrow shows the hollow stylar canal. (C) A tubular floret with five split stamens. The arrows indicate the ruptured anther walls. (D) A longitudinal section of a ray floret showing the unilocular ovary with an anatropous ovule. (E) A longitudinal section of a tubular floret showing the unilocular ovary with no ovule. (F) A longitudinal section of a tubular floret showing the oil gland cells with dense cytoplasm (arrowhead). (G) Transection of a tubular floret showing the oil gland cells (arrowheads) surrounding the style. Scale bars = 100 ␮m.

Each style of both tubular and ray florets has two stigmas and 3.2. Microsporogenesis, male gametogenesis and the formation of is of the open (or hollow) type with a circular and hollow stylar the anther wall canal (Fig. 2A–C). Every tubular floret contains five tetrasporangiate anthers, which is typical type for dicotyledonous species according At an early developmental stage, rows of archesporial cells dif- to Hu (2005) (Fig. 2B and C). The normal ovary of tubular and ray ferentiate beneath the anther epidermis, and divide periclinally to floret is unilocular with an anatropous ovule (Fig. 2D), but about form the outer primary parietal and the inner primary sporogenous 7% ovaries of the tubular florets is female sterile where ovule is cells. The archesporial cells can be recognized by their dense cyto- absent at pollen shed stage (Fig. 2E). At the base of the tubular floret plasm and conspicuous nuclei (Fig. 3A). The primary sporogenous style, oil gland cells with dense cytoplasm and conspicuous nuclei cells divide to form the secondary sporogenous cells (Fig. 3B) and surround the pistil (Fig. 2F and G). then develop into pollen mother cells (PMCs) (Fig. 3C and D). Meio- 502 Y. Deng et al. / Scientia Horticulturae 124 (2010) 500–505

Fig. 3. Microsporogenesis, male gametogenesis and the development of anther wall in tubular ﬂorets of C. multicaule. en, endothecium; ep, epidermis; ml, middle layer; pg, pollen grain; pmc, pollen mother cell; ta, tapetum; tt, tetrad. (A) A young anther showing several archesporial cells (arrowed). (B) An anther with several sporogenous cells, shortly after the differentiation of the middle layer (arrowhead) and tapetum (arrow). (C) An anther with several microsporocytes, and a developed tapetum. The immature anther wall consists of epidermis, endothecium, middle layer and tapetum (arrowed). (D–M) Various stages of meiosis in a microsporocyte. (D) Microsporocyte. (E) Prophase I. (F) Metaphase I (polar view). (G) Metaphase I (side view). (H) Anaphase I. (I) Telophase I. (J) Prophase II. (K) Metaphase II. (L) Anaphase II forming a tetrahedral tetrad. (M) Anaphase II forming a decussate tetrad. (N) Tetrahedral and decussate tetrads. (O) Microspores freshly released from a tetrad, and tapetal cells entering the anther locule. (P–T) Development in a microspore. (P) Uninucleate microspore with no vacuole. The aperture is indicated by an arrowhead. (Q) Uninucleate microspore with a large vacuole. (R) Mitotic nucleus of a microspore with a large vacuole. (S) A bicellular pollen grain with a large vegetative cell and a small generative cell. (T) A tricellular pollen grain with two sperm cells and a vegetative cell. (U) Mature pollen grain with three germinal apertures (indicated by arrowheads). (V) Radially elongated tapetal cells with two nuclei (arrowed). The arrowhead shows a mitotic anaphase in a tapetum cell. (W) Tapetal cells entering the anther locule. The arrowhead shows the tapetal periplasmodia surrounding the tetrad. The arrow shows the degenerating middle layer. (X) Once the pollen is shed, the ruptured anther wall is composed of only epidermis (indicated by arrowhead). Scale bars = 10 ␮m. sis in each PMC passes through prophase I (Fig. 3E), metaphase I but lack the vacuole (Fig. 3O). Subsequently, the exine develops and (Fig. 3F and G), anaphase I (Fig. 3H), telophase I (Fig. 3I), prophase II the germinal apertures begin to form (Fig. 3P). Later, a large vacuole (Fig. 3J), metaphase II (Fig. 3K), anaphase II (Fig. 3L) and telophase II pushes the cytoplasm and the nucleus against the wall (Fig. 3Q). (Fig. 3M), to form mostly tetrahedral, but occasionally also decus- The ﬁrst mitosis of the microspore nucleus produces two differ- sate tetrads (Fig. 3N). Thus, the cytokinesis is of the simultaneous ently sized nuclei, one of which is the vegetative, and the other is type. The newly released microspores have dense cytoplasm with the reproductive nucleus (Fig. 3R and S). The reproductive nucleus prominent and centrally placed nuclei and are irregular in shape, undergoes a mitotic division to form two sperm nuclei (Fig. 3T) Y. Deng et al. / Scientia Horticulturae 124 (2010) 500–505 503

Fig. 4. Megasporogenesis, female gametogenesis and embryogenesis in C. multicaule. ac, antipodal cells; cc, central cell; dm, degenerating megaspore; ec, egg cell; fm, functional megaspore; mmc, megaspore mother cell; n, nucleus; sc, sporogenous cell; sn, secondary nucleus; sy, synergids. (A–L) Megasporogenesis and female gametogenesis. (A) Sporogenous cell. (B) Megaspore mother cell. (C) Meiotic prophase I (indicated by arrowhead). (D) Meiotic metaphase I (indicated by arrowhead). (E) Dyad stage. (F) Tetrad stage. The functional megaspore is near the chalazal end and the other three degenerating megaspores are adjacent to the micropylar end. (G) The functional megaspore develops into a uninucleate embryo sac (arrowhead), and the remaining megaspores have degenerated (arrow). (H) Binucleate embryo sac. The arrow shows the polynucleated endothelium cells with dense cytoplasm. (I) Tetranucleate embryo sac. (J–L) Octonucleate maturing embryo sac. (J) The egg cell and one synergid. (K) The egg cell, the central cell with a secondary nucleus and two antipodal cells. (L) Another synergid and the third antipodal cell. (M–P) Embryogenesis. (M) Globular embryo stage. (N) Heart-shaped embryo stage. (O) Torpedo-shaped embryo stage. (P) Cotyledon embryo stage. Scale bars: A–L = 10 ␮m, M–P = 50 ␮m. 504 Y. Deng et al. / Scientia Horticulturae 124 (2010) 500–505 and the mature pollen grains are tricellular with three germinal Liu, 2004; Hu, 2005). Its presence in C. multicaule extends the apertures (Fig. 3U). range of dicotyledonous families where this trait is represented. The anther wall is of the glandular type, and is derived from a The liquid filled internal canal of the hollow style is surrounded parietal cell layer consisting of the epidermis, the endothecium, the by a layer of specialized inner epidermal cells, which ensure middle layer and the tapetum (Fig. 3B and C). The epidermis com- that its liquid content is withdrawn once the pistil has reached prises a single layer of cells (Fig. 3C), and is retained throughout maturity; as a result, the incoming pollen tube possibly has an gametogenesis. The middle layer is also a single layer (Fig. 3C), but easier passage through the hollow style than through the closed it gradually degenerates during the formation of the microspore, style (Hu and Zhu, 1990; Hu, 2005). Although it is likely that with only relics remaining by the time the tetrads have matured this anatomical feature facilitates seed propagation of C. multi- (Fig. 3W). The outer primary parietal cells first differentiate into caule, the full biological significance of the hollow style remains secondary parietal cells, and then divide periclinally to form the unclear. endothecium, the middle layer and the tapetum (Fig. 3B). The More than one in three of the bisexual florets of Calendula offic- tapetum cells undergo rapid mitosis, containing more than one inalis have been shown to lack an ovule, and are thus female sterile nucleus at the microsporocyte stage (Fig. 3V). They then elongate and is thought as “functionally male” (Ao, 2007). In general, the fre- and intrude into the anther locule and surround the tetrads (Fig. 3N quency of female sterile gametophytes among angiosperms lies in and W). The tapetum cells contain a high level of ploidy, simi- range about 5–15% (Teng et al., 2008), with Chrysanthemum nankin- lar to antipodal cells in the embryo sac. When the microspores gense appearing at 12% (Teng et al., 2008) and C. grandiflorum at are released from the tetrad, the tapetum cells begin to degener- 15.4% (Deng et al., 2010). In C. multicaule, the proportion of bisex- ate, so that by the time the bicellular pollen grains formed, they ual florets lacking an ovule is less than one in ten (7%), which falls have completely disintegrated and the anther wall consists only in the known range of the frequency of female sterile gameto- of endothecium. At the tricellular pollen grain stage, the endothe- phytes among angiosperms and does not significantly affect seed cium has yet to develop fibrous thickening, and the internal cells set. Therefore, whether bisexual florets lacking an ovule in C. multi- are dead. Finally, the walls rupture, allowing the pollen to disperse caule could be called as “functionally male” remains uncertain and (Figs. 3X and 2C). needs further survey. The C. multicaule archesporial cell differentiates initially to form a primary sporogenous cell, and then divides a second time to 3.3. Megasporogenesis, female gametogenesis and embryogenesis produce the PMCs. This developmental pattern is common among Chrysanthemum species (Hu, 2005). Typically for a dicotyledonous In ovary, a single sporogenous cell beneath the single layer plant, cytokinesis during meiosis is of the simultaneous type (Hu, nucellar epidermis differentiates to form the megaspore mother 2005). However, variation in the orientation of the post-division cell (MMC). At this stage, the ovule is tenuinucellate (Fig. 4A and cell plates results in the formation of both tetrahedral and decussate B). Following meiosis, the MMC forms first a dyad (Fig. 4C–E) and tetrads in C. multicaule. The morphology of the tetrads is gener- then a linear tetrad of megaspores (Fig. 4F). The megaspore near- ally believed to be species-specific, and few species exhibit such est the chalazal end is the functional gamete, while the other three polymorphism (Hu, 2005). gradually degenerate (Fig. 4F and G). The functional megaspore first The synergids usually disappear just prior to, or immediately fol- develops into a mononucleate embryo sac (Fig. 4G), then takes three lowing fertilization (Maheshwari, 1950). In an autogamous plant, mitotic divisions to develop into a binucleate (Fig. 4H), tetranucle- one synergid typically has already partially degenerated before ate (Fig. 4I) and octonucleate (Fig. 4J–L) embryo sac, successively. blooming, and will have disappeared entirely post two days of the With the opening of the floret, the two polar nuclei fuse to form a flower’s opening (Souza et al., 2002). In this study, both synergids secondary nucleus. Thus, the mature embryo sac belongs to a typ- were still present in the mature embryo sac, possibly due to the ical monosporic, eight nucleate bipolar Polygonum type according fact that C. multicaule is an allogamous species, just as is the pot to Hu (2005), consisting of an egg cell and two synergids located at marigold (Ao, 2007). micropylar end, three antipodal cells located at chalazal end, and Complete fusion occurs only after the sperm nucleus reached a large central cell with a secondary nucleus. During this process, the partially fused polar nuclei (Kapil and Bhatnagar, 1981). The the nucellar epidermis gradually degenerates and an integumen- statement was further confirmed in Sinomanglietia glauca (Xiao tal endothelium surrounds the embryo sac. The polynucleated and Yuan, 2006). In the present survey, however, they had already endothelium cells have dense cytoplasm, similar to those found in fused completely into a secondary nucleus prior to fertilization. the anther (Fig. 4H). The number of antipodal cells is always three, A similar precocious fusion has been observed in Capsella, Stipa but each cell contains different number of nucleus and usually more (Souza et al., 2002), C. officinalis (Ao, 2007) and C. grandiflorum than one (Fig. 4K and L). (Deng et al., 2010). However, the antipodal cells of C. multicaule, Upon anthesis, the zygotes undergo several divisions to form C. officinalis and C. grandiflorum do not share the same destiny. the globular proembryos by 4–6 days (Fig. 4M). It then continues to Thus, in C. multicaule and C. grandiflorum (Deng et al., 2010), they develop, first into the heart-shaped structure by 6–8 days (Fig. 4N), remain viable and multinucleate even after the formation of the egg then a torpedo-shaped one by 8–10 days (Fig. 4O). The cotyle- apparatus, whereas in C. officinalis, their degeneration is initiated don embryo is evident by days 10–12 (Fig. 4P). The time elapsed before the formation of the egg apparatus (Ao, 2007). Therefore, from blooming to seed maturity was about 3–4 weeks under our among the Asteraceae species, the antipodal cells not only vary conditions. in their size and number (Hu, 2005), but also in their timing of degeneration. 4. Discussion Species belonging to a single angiosperm family generally share the same type of embryo sac; thus, for example, among the Plant styles have been classified into three types, namely Gramineae, all embryo sacs are of the Polygonum type, as are hollow, closed and semi-closed (Knox and Williams, 1986; De those of ∼70% of all angiosperms (Maheshwari, 1950; Johri et al., Graaf et al., 2001). The hollow style, which is the predominant 1992; Reiser and Fischer, 1993). But the Asteraceae family departs type among monocotyledonous species, is rare among dicotyle- markedly from this pattern. Some species (including C. multicaule) donous species, but has been documented in a small number of form the Polygonum type (Ding et al., 1998; Ma and Wang, 2000; Papaveraceae and Aristolochiaceae species (Cresti et al., 1992; Ao, 2007; Deng et al., 2010) embryo sac, some the Adoxa type (Liu, Y. Deng et al. / Scientia Horticulturae 124 (2010) 500–505 505

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