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Floral characteristics and gametophyte development of Anemone coronaria L. and Ranunculus asiaticus L. (Ranunculaceae)
Scientia Horticulturae 138 (2012) 73–80
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Floral characteristics and gametophyte development of Anemone coronaria L. and Ranunculus asiaticus L. (Ranunculaceae) Emmy Dhooghe a,b,∗ , Wim Grunewald c,d , Dirk Reheul a , Paul Goetghebeur e , Marie-Christine Van Labeke a a
Department of Plant Production, Faculty of Bioscience Engineering, Ghent University, Coupure links 653, 9000 Gent, Belgium Plant Sciences Unit, ILVO, Institute for Agricultural and Fisheries Research, Caritasstraat 21, 9090 Melle, Belgium c Department of Plant Systems Biology, VIB, Technologiepark 927, 9052 Gent, Belgium d Department of Plant Biotechnology and Bioinformatics, Ghent University, Technologiepark 927, 9052 Gent, Belgium e Department of Biology – Spermatophytes, Faculty of Sciences, Ghent University, KL Ledeganckstraat 35, 9000 Gent, Belgium b
a r t i c l e
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Article history: Received 9 February 2011 Received in revised form 8 August 2011 Accepted 9 October 2011 Keywords: Anemone coronaria Megagametogenesis Microgametogenesis Microsporogenesis Ranunculus asiaticus Reproductive biology
a b s t r a c t Anemone coronaria and Ranunculus asiaticus both belong to the Ranunculaceae, a large plant family with many ornamentals of horticultural importance. In this study, the floral characteristics and the development of reproductive structures of these two cut flowers are investigated. On a macro-level, flower morphology was analysed by dissections, light microscopy and scanning electron microscopy (SEM). The female and male gametophyte development as well as embryogenesis was investigated on a micro-level by light microscopy of paraffin sections and fluorescence microscopy. These analyses confirmed that anthers of A. coronaria and R. asiaticus are tetrasporangiate and that the stamens of R. asiaticus can be transformed to petals. Pollen development in A. coronaria and R. asiaticus is of the simultaneous type. The mature pollen is spheroid, polyporate and bicellular. For both species, the development of the embryo sac is of the octonucleate Polygonum-type. The two polar nuclei inside the embryo sac fuse before fertilization while two of the three antipodal cells persist after fertilization. On the moment of fruitlet shedding, the embryos are not fully developed yet implicating an autonomous maturation prior to germination. Finally, pollination tests revealed that R. asiaticus ‘Alfa’ is self-incompatible. The results presented here provide new insights in the reproductive biology of A. coronaria and R. asiaticus supportive for future breeding strategies. © 2011 Elsevier B.V. All rights reserved.
1. Introduction The Ranunculaceae are a moderately large family with 59 genera and circa 2500 species (Tamura, 1995a). Most members of the Ranunculaceae are perennials and mainly herbaceous geophytes (Tamura, 1995b). Many genera of the Ranunculaceae have large and colourful flowers and are cultivated as ornamentals. Others contain pharmacological substances and are used as medicinal plants (Tamura, 1993; Hegnauer, 1995).
Abbreviations: DAPI, 4 ,6-Diamidino-2-phenylindole; SEM, Scanning electron microscopy. ∗ Corresponding author at: Department of Plant Production, Faculty of Bioscience Engineering, Ghent University, Coupure links 653, 9000 Gent, Belgium. Tel.: +32 9 264 60 71; fax: +32 9 264 62 25. E-mail addresses: [email protected] (E. Dhooghe), [email protected] (W. Grunewald), [email protected] (D. Reheul), [email protected] (P. Goetghebeur), [email protected] (M.-C. Van Labeke). 0304-4238/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.scienta.2011.10.004
The genus Anemone consists of circa 150 species distributed in temperate zones in the northern and the southern hemisphere (Tamura, 1995c). It is divided into three sections: Anemone, Anemonospermos and Pulsatilloides (Hoot and Reznicek, 1994). The section Anemone is subdivided into two groups: the Coronaria group and the Baldensis group (Hoot and Reznicek, 1994). The cultivars of Anemone coronaria belonging to the Coronaria group are important for cut flower production (Laura et al., 2006). A. coronaria is a winter-flowering perennial which flowers from February till May in the Mediterranean region (Meynet, 1993a). The genus Ranunculus consists of 600 species, making it the largest genus within the Ranunculaceae (Tamura, 1995c). Ranunculus is distributed in all continents, but mainly in non-tropical regions (Tamura, 1995c). In respect to ornamental value, Ranunculus asiaticus is by far the most cultivated Ranunculus species (Meynet, 1993b), especially in countries surrounding the Mediterranean Sea, where it is an important cut flower with blooming period ranging from February till April (Beruto and Debergh, 2004; Meynet, 1993b; Wicki-Freidl, 1988).
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Both cut flowers give the Ranunculaceae a significant economic importance. This is illustrated by the statistics of the Dutch auction with a retail of 50–60 million stalks of A. coronaria in 2010 and of 67 million stalks of R. asiaticus in 2009. Moreover this does not take the direct sell into account. Besides their cut flower importance, A. coronaria and R. asiaticus are also gaining ground as potential pot plants. Despite the large size and cosmopolitan character of the Ranunculaceae, little is known about plant morphology and floral architecture of commercial cultivars. Therefore, knowledge of the morphology of reproductive structures and the development of gametophytes and embryos is crucial when commercial cultivars are to be used in breeding research. Moreover, since the production of these plants is still based on seed, a good comprehension of the reproductive organs is necessary. To this end, this paper focuses on the morphological and reproductive characteristics of A. coronaria and R. asiaticus. 2. Materials and methods 2.1. Plant material and growth conditions Three different commercial cultivars of A. coronaria L. (grown from seed) were selected, i.e. ‘Mistral Wine’, ‘Mistral Fucsia’ (both Mistral® Line, Biancheri Creations, Camporosso Mare, Italy) and ‘Wicabri Blue’ (Wicabri® Line, the Netherlands). For R. asiaticus L., the three commercial cultivars ‘Alfa’ (Vitro Line Success® , Biancheri Creations, Camporosso Mare, Italy), ‘Krisma’ (Istituto Regionale per la Floricoltura, Sanremo, Italy) and ‘Bianco Strié’ (Biancheri Creations, Camporosso Mare, Italy) were chosen. Rhizomes were planted in September and grew until the end of April. The tuberous rhizomes were soaked in water for 24 h at 10 ◦ C and subsequently planted in a peat-mixture (pHH2 O 6.5–7.5) with 5% perlite, 5% clay and fertilizers (NPK 14-16-18, 1 kg m−3 ). The soil surface was sprayed with prochloraz (Sporgon® , 0.6 g L−1 ) directly after planting, to prevent root rot by fungi. Standard nursery practices were used for watering, fertilization and pest control. Plants were grown in a greenhouse (natural photoperiod regime) with climate condition settings of 18 ◦ C day temperature and 5 ◦ C night temperature. During the day (8 a.m. to 4 p.m.) extra assimilation light was given (assimilation light HPI-T Plus Philips, PAR: 25–30 mol m−2 s−1 ). 2.2. Macro-level flower measurements Morphological characteristics (number of sepals, tepals, petals, stamens, carpels and fruitlets) were recorded during flowering (March–April) in ten flowers per cultivar. The pollen diameter was measured of at least 20 pollen grains per cultivar by light microscopy (Olympus IX81 and imaging software CellM ). The analysed fruitlets were produced after controlled pollinations. Floral development was investigated by dissections and observed with an OLYMPUS SZX9 stereo microscope. Scanning electron microscopy (SEM) was done using a TM-1000 Tabletop microscope (HITACHI). 2.3. Micro-level flower measurements In order to study flower structure and gametophyte development, floral tissues were fixed in 1:1:18 formaldehyde:acetic acid:70% ethanol (FAA). During the subsequent five days, the plant material was gradually dehydrated using a series of solutions with decreasing ethanol and increasing butanol concentrations, resulting on the fifth day in 100% butanol solution. The sixth day, butanol was supplemented with paraffin (48 h, 50–52 ◦ C).
After solidification of the paraffin, sections of 12 m were made with a microtome (Jung AG Heidelberg). Paraffin slides were stained with fast green, safranin and haematoxylin (Johansen, 1940) and evaluated by a LEITZ GMBH Wetzlar light microscope. Pollen of the cultivars was collected randomly between 8 and 10 a.m. from February till April. 4 ,6-Diamidino-2-phenylindole (DAPI, 1.5 L mL−1 of stock solution (100 g mL−1 )) staining and fluorescence microscopy were used to study male gametophyte development. To analyse the movement of vegetative and generative nuclei, pollen was incubated on species specific germination media for 72 h at 20–22 ◦ C in dark immediately after harvest. The germination medium for A. coronaria consisted of 100 mg L−1 H3 BO3 , 700 mg L−1 Ca(NO3 )2 .4H2 O, 200 mg L−1 MgSO4 ·7H2 O, 100 mg L−1 KNO3 , 150 g L−1 PEG 6000, 0.5 g L−1 MES and a sugar content of 100 g L−1 (pH 6.0), while for R. asiaticus the medium consisted of 50 mg L−1 H3 BO3 , 300 mg L−1 CaCl2 ·2H2 O, 100 mg L−1 KH2 PO4 and a sugar content of 150 g L−1 (pH 5.5). The sperm nuclei were determined by staining the germinated pollen grains with DAPI (1.5 L mL−1 of stock solution (100 g mL−1 )) and using fluorescence microscopy (Olympus IX81). Pollinated carpels were harvested 56 h after pollination for aniline blue staining and fixed in 1:1:18 FAA for 24 h and subsequently macerated by NaOH (8 M) for 16 h. After being thoroughly washed in water, the carpels were stained in a 0.1% (w/v) aniline blue solution in 0.033 M K3 PO4 for 3 h in the dark and analysed using fluorescence microscopy (Olympus IX81). 2.4. Self-incompatibility To analyse self-compatibility in A. coronaria and R. asiaticus, seed set after self-pollination was used as a first indicator. Furthermore, an aniline blue staining was done upon selfing for all A. coronaria and R. asiaticus cultivars. The method was identical to the method mentioned in Section 2.3. 2.5. Data analysis Unless another observation number (n) is mentioned, the means described in Section 3 are based on three cultivars and ten measurements per cultivar (n = 30). The averages are presented as mean ± standard error. 3. Results 3.1. Floral macro-morphology A. coronaria has an unbranched reproductive shoot with a terminal actinomorphic flower (Fig. 1A). Three to five bracts, sessile or sometimes slightly connate at the base, form an involucre (Fig. 1B). The bracts have an entire margin at the base and are dentate to even lobed at the top. Occasionally the bracts can be petaloid (Fig. 1C). The circa nine tepals are petaloid and obovate (mean number: 9.3 ± 0.4). The stamens are numerous (mean number: 336.3 ± 12.4) with centripetal development (Fig. 1D). Young stamens and carpels are very similar (Fig. 1D inset). Using SEM it could be shown that the pollen is released from the anthers by a longitudinal slit (Fig. 1E). The free, mature pollen grains are spheroid and polyporate (Fig. 1F), 31.6 ± 0.3 m in diameter (n = 456). The circa 1000 uni-ovular carpels (mean number: 996.8 ± 86.6) have a unicellular-papillate dry stigma (Fig. 1G–I) and are arranged on a conspicuously convex receptacle. The receptacle enlarges after anthesis and continues to enlarge during fruitlet formation. The fruitlets or achenes of all three cultivars of A. coronaria, obtained by pollination within the cultivar (mean number: 728.4 ± 70.7), are characterised by long soft curled hairs (Fig. 1J).
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Fig. 1. Macro-level flower characteristics of Anemone coronaria. (A) Unbranched pedicel of ‘Wicabri Blue’. (B) The bracts form an involucre. (C) Petaloid bract of ‘Mistral Fucsia’. (D) The stamens mature centripetally (‘Wicabri Blue’). By flower dissections the centripetal maturation of the stamens was noticed (inset) (‘Mistral Wine’). (E) Scanning electron micrograph of an anther of ‘Mistral Fucsia’, which releases the pollen by a longitudinal slit. (F) Pollen of ‘Wicabri Blue’ is spheroid and polyporate (SEM). (G) Longitudinal section of the stigma with unicellular papillae (‘Mistral Fucsia’). (H) SEM gives a detailed view on the papillae (‘Mistral Fucsia’). (I) The carpels are uni-ovular (‘Mistral Fucsia’). (J) The fruitlets (= achenes) of ‘Mistral Fucsia’ with soft curled hairs. Scale bars: (A–C) = 5 mm; (D) = 10 mm; (D inset) = 250 m; (E) = 300 m; (F) = 30 m; (G and H) = 60 m; (I and J) = 1 mm.
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Fig. 2. Macro-level flower characteristics of Ranunculus asiaticus. (A and B) A flower of ‘Alfa’ with separate sepals and petals. (C) The petals have a rudimentary nectary pit. Inset: close-up view of basal part of petal (‘Alfa’). (D) In early stages, the stamens and carpels look similar (‘Alfa’). (E) Scanning electron micrograph of the transformation of stamens to petals or intermediate floral organs (‘Krisma’). Inset: light microscopy of a transformed stamen (‘Alfa’). (F) Scanning electron micrograph of an anther of ‘Bianco Strié’. (G) Pollen of ‘Alfa’ is spheroid and polyporate (SEM). (H) Scanning electron microscopic view on the papillate stigma of ‘Krisma’. (I) The carpels are uni-ovular (‘Krisma’). (J) The carpels/fruitlets of ‘Bianco Strié’ sit on a well-developed receptacle, which expands during flowering and maturation. Inset: the fruitlets or achenes are winged (‘Bianco Strié’). Scale bars: (A and B) = 5 mm; (C and J) = 10 mm; (C inset and J inset) = 1 mm; (D, E inset and H) = 200 m; (E, F, and I) = 1 mm; (G) = 30 m.
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Fig. 3. Male gametophyte development of Anemone coronaria and Ranunculus asiaticus. (A) Transverse paraffin sections of anthers in a small flower bud of Anemone coronaria ‘Mistral Fucsia’ shows that they are tetrasporangiate. The anther has two thecae. (B) Later flower stage of Anemone coronaria ‘Mistral Wine’ (flower bud is coloured but still closed and is in a straight line with pedicel) with pollen sacs and microspores. The anther is still divided in four parts by the connective and the thecal septa. (C) Detail of pollen sac and anther wall layers of Anemone coronaria ‘Mistral Wine’, showing epidermis (ep), endothecium (en), two middle layers (ml) and tapetum (ta). (D) A pollen mother cell of Anemone coronaria ‘Mistral Fucsia’. (E) A tetrad of Anemone coronaria ‘Mistral Fucsia’. Inset: close-up view of a tetrad showing the chromosomes in Ranunculus asiaticus ‘Bianco Strié’. (F) A microspore with a central nucleus of Anemone coronaria ‘Mistral Fucsia’. (G) A microspore with a lateral nucleus of Anemone coronaria ‘Mistral Fucsia’. (H) A mature bicellular pollen grain of Anemone coronaria ‘Mistral Fucsia’. The vegetative nucleus is round, while the generative nucleus is more or less sickle-shaped. Scale bars: (A–C) = 100 m; (D–H) = 20 m.
Fig. 4. Vegetative and generative nucleus movement in pollen tubes of Anemone coronaria and Ranunculus asiaticus. In the pollen tube, the generative nucleus becomes more rounded and the vegetative nucleus has an irregular shape. (A) The vegetative nucleus precedes the generative nucleus in a pollen tube of Ranunculus asiaticus ‘Bianco Strié’. Inset: close-up of migrating nuclei. (B) The generative nucleus joins the vegetative nucleus in a pollen tube of Ranunculus asiaticus ‘Bianco Strié’. Inset: close-up of migrating nuclei. (C) The vegetative nucleus lags behind the generative nucleus in Anemone coronaria ‘Mistral Fucsia’. Inset: close-up of migrating nuclei. (D) The generative nucleus of a pollen tube of Anemone coronaria ‘Mistral Fucsia’ is divided in two sperm nuclei and is behind the vegetative nucleus. (E) A pollen tube of Anemone coronaria ‘Mistral Fucsia’ with two gametes preceding the vegetative nucleus. (F) Occasionally the vegetative nucleus was in between two gametes (Ranunculus asiaticus ‘Bianco Strié’). Arrowheads point to the corresponding pollen grains. Scale bars: (A–F) = 100 m.
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Fig. 5. Carpel and female gametophyte development in Ranunculus asiaticus ‘Bianco Strié’. Megagametogenesis is according to the Polygonum-type. (A) Longitudinal sections of a young receptacle. Carpels are maturing acropetally. (B) A functional megaspore. (C) The first mitosis of the megaspore results in a micropylar and chalazal nucleus indicated by two black arrowheads. (D) The mature female gametophyte is octonuclear: two polar nuclei in the middle of the central cell, two synergids (grey arrowheads) and one egg cell (white arrowhead) and three antipodal cells (black arrows). Scale bars: (A) = 1 mm; (B–D) = 100 m.
R. asiaticus has thyrsoids as inflorescences; each cyme has one to five flowers. The flowers are actinomorphic but in contrast to A. coronaria, R. asiaticus has 4–8 ovate sepals and circa 120 obovate petals (mean number: 117.2 ± 8.6) (Fig. 2A and B). The studied cultivars of R. asiaticus show rudimentary nectaries at the base of the petals (Fig. 2C). The number of stamens is highly variable in the studied cultivars (mean number: 36.7 ± 5.9). As Anemone, the young stamens and carpels are very similar (Fig. 2D). Often, some of the stamens develop into petals. This results in petals with stamen remnants or in intermediate floral organs (Fig. 2E). Spheroid and polyporate pollen grains are released from the anthers by a longitudinal slit (Fig. 2F). The pollen grain diameter is 27.9 ± 0.4 m (n = 111) (Fig. 2G). The carpels are uni-ovular (Fig. 2I) and have a unicellular-papillate dry stigma (Fig. 2H) while the style is solid. The numerous carpels (mean number: 663.0 ± 38.1) are arranged on a well-developed receptacle, which expands during flowering (Fig. 2J). The fruitlets are winged achenes (Fig. 2J inset). The cultivar ‘Bianco Strié’ produced 493.0 ± 63.6 (n = 10) fruitlets after pollination within the cultivar. No fruitlets for ‘Alfa’ were produced after pollination with pollen of the same (clonal) cultivar. Concerning ‘Krisma’, the fruitlets could not be counted: due to an insufficient number of stamens not enough pollen needed for an efficient pollination could be collected.
3.2. Floral micro-morphology 3.2.1. Male gametophyte development Transverse paraffin sectioning showed that the anthers of A. coronaria and R. asiaticus are tetrasporangiate (Fig. 3A and B), each sporangium with epidermis, endothecium, two middle layers and a tapetum layer (Fig. 3C). At the earliest stages studied, the male reproductive cells were at the stage of pollen mother cells (Fig. 3D). A. coronaria and R. asiaticus belong to the simultaneous group, in which the first meiotic division is directly followed by a second division, yielding a tetrad of microspores (Fig. 3E). In a next step the microspores with a central nucleus are released (Fig. 3F). Subsequently, the nucleus moves to the margin of the cell (Fig. 3G), whereafter the nucleus divides into a large rounded vegetative nucleus and a clear sickle-shaped generative nucleus (Fig. 3H). After entering the pollen tube, the generative nucleus becomes more rounded, while the vegetative nucleus is often irregular in shape. From this moment on, the position of both nuclei to one another can change. Sometimes the generative nucleus is behind the vegetative nucleus (Fig. 4A). In other cases the generative nucleus is catching up with the vegetative nucleus (Fig. 4B) or can even be in front of the latter (Fig. 4C). As a consequence the position
Fig. 6. Embryogenesis of Anemone coronaria. (A) The embryo sac of Anemone coronaria ‘Mistral Fucsia’ has large antipodal cells (a) which persist long after fertilization. The embryo (e) is already in the dermatogen stage. The prominent nucleus in the centre is derived from the fusion of the two polar nuclei and the sperm cell. (B) A torpedo-shaped embryo of Anemone coronaria ‘Mistral Fucsia’ in a fruitlet after shedding (nine weeks after pollination). Scale bars: (A) = 100 m and (B) = 250 m.
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Fig. 7. Pollen tubes visualised by aniline blue staining 56 h after pollination. Inhibited and twisted pollen tube growth in the styles of self-pollinated Ranunculus asiaticus ‘Alfa’. Scale bar = 100 m.
of the two sperm nuclei (obtained after division of the generative nucleus or the second pollen mitosis) can be behind or in front of the vegetative nucleus (Fig. 4D and E). Sporadically the vegetative nucleus could be observed in between the two sperm nuclei or moving together with the sperm nuclei (Fig. 4F). 3.2.2. Female gametophyte development and embryogenesis Longitudinal paraffin sections of the receptacle of A. coronaria and R. asiaticus showed that carpels are maturing from the base of the receptacle upwards (Fig. 5A). The embryo sac development of both A. coronaria and R. asiaticus follows the Polygonum-type and the mature female gametophyte is octonucleate (Fig. 5B–D). The embryo sacs of both A. coronaria and R. asiaticus are characterised by two typical features: (1) the presence of the large antipodal cells of which two of the three persist long after fertilization and (2) the prominent nucleus of the central cell, which is derived from the fused two polar nuclei, long before fertilization (Fig. 6A). When the fruitlets are ripe and are shed, the embryos are not yet fully developed; their growth is arrested at the torpedo stage (Fig. 6B). 3.3. Self-incompatibility Because R. asiaticus ‘Krisma’ did not produce enough pollen due to a suboptimal number of stamens, seed set could not be analysed. The remaining tested cultivars are all self-compatible based on adequate seed set except for R. asiaticus ‘Alfa’, which did not result in fruitlets, indicating self-incompatibility. To confirm the putative self-incompatibility of ‘Alfa’, in vivo pollen tube growth was tracked by aniline blue staining upon selfing. Because only a limited amount of pollen is needed for this purpose, all A. coronaria and R. asiaticus cultivars could be analysed. Of all these cultivars, only in the case of R. asiaticus ‘Alfa’ pollen tube growth in the stylar region was strongly inhibited (Fig. 7), confirming that ‘Alfa’ is largely self-incompatible. 4. Discussion In line with previous reports (Endress and Igersheim, 1999; Ren et al., 2009, 2010; Tamura, 1995c; Wang and Ren, 2008), the morphological analyses reported in this study clearly demonstrate
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the heterogeneity within the Ranunculaceae family. In the Ranunculaceae, there are four perianth types: morphologically distinct petaloid sepals and petals (e.g., Aquilegia), only petaloid tepals (e.g., Anemone), green sepals and coloured petals (e.g., Ranunculus) and green sepals and no petals (e.g., Beesia, Trautvetteria) (Kramer, 2009; Tamura, 1995c). A. coronaria has petaloid tepals, while R. asiaticus has both sepals and petals. A. coronaria has no nectaries and the studied cultivars of R. asiaticus show only rudimentary nectaries. This is in contrast with Trollius, Nigella and other Ranunculus species in which nectaries were observed which are in a pit or a pocket near the base of the petal (Tamura, 1995d). The studied cultivars of R. asiaticus showed a variable number of stamens. As both stamens and petals are arranged on the same spiral, a possible explanation is that stamens and petals can be converted in one another during tissue differentiation (Ronse Decraene and Smets, 1995; Tamura, 1995d). Indeed, early stages of petals, stamens and even carpels are hardly distinguishable. This is in line with previous studies on Anemone rivularis (Chang et al., 2005) and other studies on members of the Ranunculaceae (Ren et al., 2009). In this study, SEM confirmed the conversion of stamens to petals in R. asiaticus. Regarding gametophyte development and embryogenesis, the two genera go through similar developmental stages resulting in not fully developed embryos at the time of fruitlet shedding: the embryogenic growth is arrested at the torpedo stage. This was also observed in Helleborus niger (Niimi et al., 2006) and Hepatica nobilis (Nomizu et al., 2004), both members of the Ranunculaceae. The Ranunculus cultivar ‘Alfa’ is self-incompatible: although the pollen was able to germinate, its tube growth was arrested in the style, suggesting gametophytic self-incompatibility. This is in line with previous reports showing gametophytic self-incompatibility in some Ranunculus species (Horovitz, 1985; Lundqvist, 1990). 5. Conclusions The flower morphology of A. coronaria and R. asiaticus differs in some aspects (e.g., perianth type, nectaries). R. asiaticus showed a non conserved number of stamens, which can be explained by the conversion of stamens to petals. Seeds contain underdeveloped embryos and thus maturation is needed prior to germination. This maturation process is often accomplished by a cold treatment. For the Ranunculus cultivar ‘Alfa’ also self-incompatibility was noticed. All these characteristics are important for propagation of and breeding research using these ornamentals. Acknowledgements The authors thank T. Versluys, C. Petit and B. Meersman for technical support. Istituto Regionale per la Floricoltura and Biancheri Creations are acknowledged for supplying the rhizomes of Anemone coronaria and Ranunculus asiaticus. This work was supported by Research Foundation Flanders (FWO) to W.G. References Beruto, M., Debergh, P., 2004. Micropropagation of Ranunculus asiaticus: a review and perspectives. Plant Cell Tissue Organ Cult. 77, 221–230. Chang, H.L., Ren, Y., Lu, A.M., 2005. Floral morphogenesis of Anemone rivularis BuchHam. ex DC. var. flore-minore Maxim. (Ranunculaceae) with special emphasis on androecium developmental sequence. J. Integr. Plant Biol. 47, 257–263. Endress, P.K., Igersheim, A., 1999. Gynoecium diversity and systematics of the basal eudicots. Bot. J. Linn. Soc. 130, 305–393. Hegnauer, V.R., 1995. Vergleichende phytochemie und chemotaxonomie. In: Engler, A., Prantl, K. (Eds.), Die Natürlichen Pflanzenfamilien. Bd. 17 a IV Angiospermae. Ordnung Ranunculales. Fam. Ranunculaceae. Duncker & Humblot, Berlin, pp. 185–210. Horovitz, A., 1985. Ranunculus. In: Halevy, A.H. (Ed.), CRC Handbook of Flowering. CRC Press, Florida, pp. 155–161. Hoot, S.B., Reznicek, A., 1994. Phylogenetic relationships in Anemone (Ranunculaceae) based on morphology and chloroplast DNA. Syst. Bot. 19, 169–200.
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Floral characteristics and gametophyte development of Anemone coronaria L. and Ranunculus asiaticus L. (Ranunculaceae) Emmy Dhooghe a,b,∗ , Wim Grunewald c,d , Dirk Reheul a , Paul Goetghebeur e , Marie-Christine Van Labeke a a
Department of Plant Production, Faculty of Bioscience Engineering, Ghent University, Coupure links 653, 9000 Gent, Belgium Plant Sciences Unit, ILVO, Institute for Agricultural and Fisheries Research, Caritasstraat 21, 9090 Melle, Belgium c Department of Plant Systems Biology, VIB, Technologiepark 927, 9052 Gent, Belgium d Department of Plant Biotechnology and Bioinformatics, Ghent University, Technologiepark 927, 9052 Gent, Belgium e Department of Biology – Spermatophytes, Faculty of Sciences, Ghent University, KL Ledeganckstraat 35, 9000 Gent, Belgium b
a r t i c l e
i n f o
Article history: Received 9 February 2011 Received in revised form 8 August 2011 Accepted 9 October 2011 Keywords: Anemone coronaria Megagametogenesis Microgametogenesis Microsporogenesis Ranunculus asiaticus Reproductive biology
a b s t r a c t Anemone coronaria and Ranunculus asiaticus both belong to the Ranunculaceae, a large plant family with many ornamentals of horticultural importance. In this study, the floral characteristics and the development of reproductive structures of these two cut flowers are investigated. On a macro-level, flower morphology was analysed by dissections, light microscopy and scanning electron microscopy (SEM). The female and male gametophyte development as well as embryogenesis was investigated on a micro-level by light microscopy of paraffin sections and fluorescence microscopy. These analyses confirmed that anthers of A. coronaria and R. asiaticus are tetrasporangiate and that the stamens of R. asiaticus can be transformed to petals. Pollen development in A. coronaria and R. asiaticus is of the simultaneous type. The mature pollen is spheroid, polyporate and bicellular. For both species, the development of the embryo sac is of the octonucleate Polygonum-type. The two polar nuclei inside the embryo sac fuse before fertilization while two of the three antipodal cells persist after fertilization. On the moment of fruitlet shedding, the embryos are not fully developed yet implicating an autonomous maturation prior to germination. Finally, pollination tests revealed that R. asiaticus ‘Alfa’ is self-incompatible. The results presented here provide new insights in the reproductive biology of A. coronaria and R. asiaticus supportive for future breeding strategies. © 2011 Elsevier B.V. All rights reserved.
1. Introduction The Ranunculaceae are a moderately large family with 59 genera and circa 2500 species (Tamura, 1995a). Most members of the Ranunculaceae are perennials and mainly herbaceous geophytes (Tamura, 1995b). Many genera of the Ranunculaceae have large and colourful flowers and are cultivated as ornamentals. Others contain pharmacological substances and are used as medicinal plants (Tamura, 1993; Hegnauer, 1995).
Abbreviations: DAPI, 4 ,6-Diamidino-2-phenylindole; SEM, Scanning electron microscopy. ∗ Corresponding author at: Department of Plant Production, Faculty of Bioscience Engineering, Ghent University, Coupure links 653, 9000 Gent, Belgium. Tel.: +32 9 264 60 71; fax: +32 9 264 62 25. E-mail addresses: [email protected] (E. Dhooghe), [email protected] (W. Grunewald), [email protected] (D. Reheul), [email protected] (P. Goetghebeur), [email protected] (M.-C. Van Labeke). 0304-4238/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.scienta.2011.10.004
The genus Anemone consists of circa 150 species distributed in temperate zones in the northern and the southern hemisphere (Tamura, 1995c). It is divided into three sections: Anemone, Anemonospermos and Pulsatilloides (Hoot and Reznicek, 1994). The section Anemone is subdivided into two groups: the Coronaria group and the Baldensis group (Hoot and Reznicek, 1994). The cultivars of Anemone coronaria belonging to the Coronaria group are important for cut flower production (Laura et al., 2006). A. coronaria is a winter-flowering perennial which flowers from February till May in the Mediterranean region (Meynet, 1993a). The genus Ranunculus consists of 600 species, making it the largest genus within the Ranunculaceae (Tamura, 1995c). Ranunculus is distributed in all continents, but mainly in non-tropical regions (Tamura, 1995c). In respect to ornamental value, Ranunculus asiaticus is by far the most cultivated Ranunculus species (Meynet, 1993b), especially in countries surrounding the Mediterranean Sea, where it is an important cut flower with blooming period ranging from February till April (Beruto and Debergh, 2004; Meynet, 1993b; Wicki-Freidl, 1988).
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Both cut flowers give the Ranunculaceae a significant economic importance. This is illustrated by the statistics of the Dutch auction with a retail of 50–60 million stalks of A. coronaria in 2010 and of 67 million stalks of R. asiaticus in 2009. Moreover this does not take the direct sell into account. Besides their cut flower importance, A. coronaria and R. asiaticus are also gaining ground as potential pot plants. Despite the large size and cosmopolitan character of the Ranunculaceae, little is known about plant morphology and floral architecture of commercial cultivars. Therefore, knowledge of the morphology of reproductive structures and the development of gametophytes and embryos is crucial when commercial cultivars are to be used in breeding research. Moreover, since the production of these plants is still based on seed, a good comprehension of the reproductive organs is necessary. To this end, this paper focuses on the morphological and reproductive characteristics of A. coronaria and R. asiaticus. 2. Materials and methods 2.1. Plant material and growth conditions Three different commercial cultivars of A. coronaria L. (grown from seed) were selected, i.e. ‘Mistral Wine’, ‘Mistral Fucsia’ (both Mistral® Line, Biancheri Creations, Camporosso Mare, Italy) and ‘Wicabri Blue’ (Wicabri® Line, the Netherlands). For R. asiaticus L., the three commercial cultivars ‘Alfa’ (Vitro Line Success® , Biancheri Creations, Camporosso Mare, Italy), ‘Krisma’ (Istituto Regionale per la Floricoltura, Sanremo, Italy) and ‘Bianco Strié’ (Biancheri Creations, Camporosso Mare, Italy) were chosen. Rhizomes were planted in September and grew until the end of April. The tuberous rhizomes were soaked in water for 24 h at 10 ◦ C and subsequently planted in a peat-mixture (pHH2 O 6.5–7.5) with 5% perlite, 5% clay and fertilizers (NPK 14-16-18, 1 kg m−3 ). The soil surface was sprayed with prochloraz (Sporgon® , 0.6 g L−1 ) directly after planting, to prevent root rot by fungi. Standard nursery practices were used for watering, fertilization and pest control. Plants were grown in a greenhouse (natural photoperiod regime) with climate condition settings of 18 ◦ C day temperature and 5 ◦ C night temperature. During the day (8 a.m. to 4 p.m.) extra assimilation light was given (assimilation light HPI-T Plus Philips, PAR: 25–30 mol m−2 s−1 ). 2.2. Macro-level flower measurements Morphological characteristics (number of sepals, tepals, petals, stamens, carpels and fruitlets) were recorded during flowering (March–April) in ten flowers per cultivar. The pollen diameter was measured of at least 20 pollen grains per cultivar by light microscopy (Olympus IX81 and imaging software CellM ). The analysed fruitlets were produced after controlled pollinations. Floral development was investigated by dissections and observed with an OLYMPUS SZX9 stereo microscope. Scanning electron microscopy (SEM) was done using a TM-1000 Tabletop microscope (HITACHI). 2.3. Micro-level flower measurements In order to study flower structure and gametophyte development, floral tissues were fixed in 1:1:18 formaldehyde:acetic acid:70% ethanol (FAA). During the subsequent five days, the plant material was gradually dehydrated using a series of solutions with decreasing ethanol and increasing butanol concentrations, resulting on the fifth day in 100% butanol solution. The sixth day, butanol was supplemented with paraffin (48 h, 50–52 ◦ C).
After solidification of the paraffin, sections of 12 m were made with a microtome (Jung AG Heidelberg). Paraffin slides were stained with fast green, safranin and haematoxylin (Johansen, 1940) and evaluated by a LEITZ GMBH Wetzlar light microscope. Pollen of the cultivars was collected randomly between 8 and 10 a.m. from February till April. 4 ,6-Diamidino-2-phenylindole (DAPI, 1.5 L mL−1 of stock solution (100 g mL−1 )) staining and fluorescence microscopy were used to study male gametophyte development. To analyse the movement of vegetative and generative nuclei, pollen was incubated on species specific germination media for 72 h at 20–22 ◦ C in dark immediately after harvest. The germination medium for A. coronaria consisted of 100 mg L−1 H3 BO3 , 700 mg L−1 Ca(NO3 )2 .4H2 O, 200 mg L−1 MgSO4 ·7H2 O, 100 mg L−1 KNO3 , 150 g L−1 PEG 6000, 0.5 g L−1 MES and a sugar content of 100 g L−1 (pH 6.0), while for R. asiaticus the medium consisted of 50 mg L−1 H3 BO3 , 300 mg L−1 CaCl2 ·2H2 O, 100 mg L−1 KH2 PO4 and a sugar content of 150 g L−1 (pH 5.5). The sperm nuclei were determined by staining the germinated pollen grains with DAPI (1.5 L mL−1 of stock solution (100 g mL−1 )) and using fluorescence microscopy (Olympus IX81). Pollinated carpels were harvested 56 h after pollination for aniline blue staining and fixed in 1:1:18 FAA for 24 h and subsequently macerated by NaOH (8 M) for 16 h. After being thoroughly washed in water, the carpels were stained in a 0.1% (w/v) aniline blue solution in 0.033 M K3 PO4 for 3 h in the dark and analysed using fluorescence microscopy (Olympus IX81). 2.4. Self-incompatibility To analyse self-compatibility in A. coronaria and R. asiaticus, seed set after self-pollination was used as a first indicator. Furthermore, an aniline blue staining was done upon selfing for all A. coronaria and R. asiaticus cultivars. The method was identical to the method mentioned in Section 2.3. 2.5. Data analysis Unless another observation number (n) is mentioned, the means described in Section 3 are based on three cultivars and ten measurements per cultivar (n = 30). The averages are presented as mean ± standard error. 3. Results 3.1. Floral macro-morphology A. coronaria has an unbranched reproductive shoot with a terminal actinomorphic flower (Fig. 1A). Three to five bracts, sessile or sometimes slightly connate at the base, form an involucre (Fig. 1B). The bracts have an entire margin at the base and are dentate to even lobed at the top. Occasionally the bracts can be petaloid (Fig. 1C). The circa nine tepals are petaloid and obovate (mean number: 9.3 ± 0.4). The stamens are numerous (mean number: 336.3 ± 12.4) with centripetal development (Fig. 1D). Young stamens and carpels are very similar (Fig. 1D inset). Using SEM it could be shown that the pollen is released from the anthers by a longitudinal slit (Fig. 1E). The free, mature pollen grains are spheroid and polyporate (Fig. 1F), 31.6 ± 0.3 m in diameter (n = 456). The circa 1000 uni-ovular carpels (mean number: 996.8 ± 86.6) have a unicellular-papillate dry stigma (Fig. 1G–I) and are arranged on a conspicuously convex receptacle. The receptacle enlarges after anthesis and continues to enlarge during fruitlet formation. The fruitlets or achenes of all three cultivars of A. coronaria, obtained by pollination within the cultivar (mean number: 728.4 ± 70.7), are characterised by long soft curled hairs (Fig. 1J).
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Fig. 1. Macro-level flower characteristics of Anemone coronaria. (A) Unbranched pedicel of ‘Wicabri Blue’. (B) The bracts form an involucre. (C) Petaloid bract of ‘Mistral Fucsia’. (D) The stamens mature centripetally (‘Wicabri Blue’). By flower dissections the centripetal maturation of the stamens was noticed (inset) (‘Mistral Wine’). (E) Scanning electron micrograph of an anther of ‘Mistral Fucsia’, which releases the pollen by a longitudinal slit. (F) Pollen of ‘Wicabri Blue’ is spheroid and polyporate (SEM). (G) Longitudinal section of the stigma with unicellular papillae (‘Mistral Fucsia’). (H) SEM gives a detailed view on the papillae (‘Mistral Fucsia’). (I) The carpels are uni-ovular (‘Mistral Fucsia’). (J) The fruitlets (= achenes) of ‘Mistral Fucsia’ with soft curled hairs. Scale bars: (A–C) = 5 mm; (D) = 10 mm; (D inset) = 250 m; (E) = 300 m; (F) = 30 m; (G and H) = 60 m; (I and J) = 1 mm.
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Fig. 2. Macro-level flower characteristics of Ranunculus asiaticus. (A and B) A flower of ‘Alfa’ with separate sepals and petals. (C) The petals have a rudimentary nectary pit. Inset: close-up view of basal part of petal (‘Alfa’). (D) In early stages, the stamens and carpels look similar (‘Alfa’). (E) Scanning electron micrograph of the transformation of stamens to petals or intermediate floral organs (‘Krisma’). Inset: light microscopy of a transformed stamen (‘Alfa’). (F) Scanning electron micrograph of an anther of ‘Bianco Strié’. (G) Pollen of ‘Alfa’ is spheroid and polyporate (SEM). (H) Scanning electron microscopic view on the papillate stigma of ‘Krisma’. (I) The carpels are uni-ovular (‘Krisma’). (J) The carpels/fruitlets of ‘Bianco Strié’ sit on a well-developed receptacle, which expands during flowering and maturation. Inset: the fruitlets or achenes are winged (‘Bianco Strié’). Scale bars: (A and B) = 5 mm; (C and J) = 10 mm; (C inset and J inset) = 1 mm; (D, E inset and H) = 200 m; (E, F, and I) = 1 mm; (G) = 30 m.
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Fig. 3. Male gametophyte development of Anemone coronaria and Ranunculus asiaticus. (A) Transverse paraffin sections of anthers in a small flower bud of Anemone coronaria ‘Mistral Fucsia’ shows that they are tetrasporangiate. The anther has two thecae. (B) Later flower stage of Anemone coronaria ‘Mistral Wine’ (flower bud is coloured but still closed and is in a straight line with pedicel) with pollen sacs and microspores. The anther is still divided in four parts by the connective and the thecal septa. (C) Detail of pollen sac and anther wall layers of Anemone coronaria ‘Mistral Wine’, showing epidermis (ep), endothecium (en), two middle layers (ml) and tapetum (ta). (D) A pollen mother cell of Anemone coronaria ‘Mistral Fucsia’. (E) A tetrad of Anemone coronaria ‘Mistral Fucsia’. Inset: close-up view of a tetrad showing the chromosomes in Ranunculus asiaticus ‘Bianco Strié’. (F) A microspore with a central nucleus of Anemone coronaria ‘Mistral Fucsia’. (G) A microspore with a lateral nucleus of Anemone coronaria ‘Mistral Fucsia’. (H) A mature bicellular pollen grain of Anemone coronaria ‘Mistral Fucsia’. The vegetative nucleus is round, while the generative nucleus is more or less sickle-shaped. Scale bars: (A–C) = 100 m; (D–H) = 20 m.
Fig. 4. Vegetative and generative nucleus movement in pollen tubes of Anemone coronaria and Ranunculus asiaticus. In the pollen tube, the generative nucleus becomes more rounded and the vegetative nucleus has an irregular shape. (A) The vegetative nucleus precedes the generative nucleus in a pollen tube of Ranunculus asiaticus ‘Bianco Strié’. Inset: close-up of migrating nuclei. (B) The generative nucleus joins the vegetative nucleus in a pollen tube of Ranunculus asiaticus ‘Bianco Strié’. Inset: close-up of migrating nuclei. (C) The vegetative nucleus lags behind the generative nucleus in Anemone coronaria ‘Mistral Fucsia’. Inset: close-up of migrating nuclei. (D) The generative nucleus of a pollen tube of Anemone coronaria ‘Mistral Fucsia’ is divided in two sperm nuclei and is behind the vegetative nucleus. (E) A pollen tube of Anemone coronaria ‘Mistral Fucsia’ with two gametes preceding the vegetative nucleus. (F) Occasionally the vegetative nucleus was in between two gametes (Ranunculus asiaticus ‘Bianco Strié’). Arrowheads point to the corresponding pollen grains. Scale bars: (A–F) = 100 m.
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Fig. 5. Carpel and female gametophyte development in Ranunculus asiaticus ‘Bianco Strié’. Megagametogenesis is according to the Polygonum-type. (A) Longitudinal sections of a young receptacle. Carpels are maturing acropetally. (B) A functional megaspore. (C) The first mitosis of the megaspore results in a micropylar and chalazal nucleus indicated by two black arrowheads. (D) The mature female gametophyte is octonuclear: two polar nuclei in the middle of the central cell, two synergids (grey arrowheads) and one egg cell (white arrowhead) and three antipodal cells (black arrows). Scale bars: (A) = 1 mm; (B–D) = 100 m.
R. asiaticus has thyrsoids as inflorescences; each cyme has one to five flowers. The flowers are actinomorphic but in contrast to A. coronaria, R. asiaticus has 4–8 ovate sepals and circa 120 obovate petals (mean number: 117.2 ± 8.6) (Fig. 2A and B). The studied cultivars of R. asiaticus show rudimentary nectaries at the base of the petals (Fig. 2C). The number of stamens is highly variable in the studied cultivars (mean number: 36.7 ± 5.9). As Anemone, the young stamens and carpels are very similar (Fig. 2D). Often, some of the stamens develop into petals. This results in petals with stamen remnants or in intermediate floral organs (Fig. 2E). Spheroid and polyporate pollen grains are released from the anthers by a longitudinal slit (Fig. 2F). The pollen grain diameter is 27.9 ± 0.4 m (n = 111) (Fig. 2G). The carpels are uni-ovular (Fig. 2I) and have a unicellular-papillate dry stigma (Fig. 2H) while the style is solid. The numerous carpels (mean number: 663.0 ± 38.1) are arranged on a well-developed receptacle, which expands during flowering (Fig. 2J). The fruitlets are winged achenes (Fig. 2J inset). The cultivar ‘Bianco Strié’ produced 493.0 ± 63.6 (n = 10) fruitlets after pollination within the cultivar. No fruitlets for ‘Alfa’ were produced after pollination with pollen of the same (clonal) cultivar. Concerning ‘Krisma’, the fruitlets could not be counted: due to an insufficient number of stamens not enough pollen needed for an efficient pollination could be collected.
3.2. Floral micro-morphology 3.2.1. Male gametophyte development Transverse paraffin sectioning showed that the anthers of A. coronaria and R. asiaticus are tetrasporangiate (Fig. 3A and B), each sporangium with epidermis, endothecium, two middle layers and a tapetum layer (Fig. 3C). At the earliest stages studied, the male reproductive cells were at the stage of pollen mother cells (Fig. 3D). A. coronaria and R. asiaticus belong to the simultaneous group, in which the first meiotic division is directly followed by a second division, yielding a tetrad of microspores (Fig. 3E). In a next step the microspores with a central nucleus are released (Fig. 3F). Subsequently, the nucleus moves to the margin of the cell (Fig. 3G), whereafter the nucleus divides into a large rounded vegetative nucleus and a clear sickle-shaped generative nucleus (Fig. 3H). After entering the pollen tube, the generative nucleus becomes more rounded, while the vegetative nucleus is often irregular in shape. From this moment on, the position of both nuclei to one another can change. Sometimes the generative nucleus is behind the vegetative nucleus (Fig. 4A). In other cases the generative nucleus is catching up with the vegetative nucleus (Fig. 4B) or can even be in front of the latter (Fig. 4C). As a consequence the position
Fig. 6. Embryogenesis of Anemone coronaria. (A) The embryo sac of Anemone coronaria ‘Mistral Fucsia’ has large antipodal cells (a) which persist long after fertilization. The embryo (e) is already in the dermatogen stage. The prominent nucleus in the centre is derived from the fusion of the two polar nuclei and the sperm cell. (B) A torpedo-shaped embryo of Anemone coronaria ‘Mistral Fucsia’ in a fruitlet after shedding (nine weeks after pollination). Scale bars: (A) = 100 m and (B) = 250 m.
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Fig. 7. Pollen tubes visualised by aniline blue staining 56 h after pollination. Inhibited and twisted pollen tube growth in the styles of self-pollinated Ranunculus asiaticus ‘Alfa’. Scale bar = 100 m.
of the two sperm nuclei (obtained after division of the generative nucleus or the second pollen mitosis) can be behind or in front of the vegetative nucleus (Fig. 4D and E). Sporadically the vegetative nucleus could be observed in between the two sperm nuclei or moving together with the sperm nuclei (Fig. 4F). 3.2.2. Female gametophyte development and embryogenesis Longitudinal paraffin sections of the receptacle of A. coronaria and R. asiaticus showed that carpels are maturing from the base of the receptacle upwards (Fig. 5A). The embryo sac development of both A. coronaria and R. asiaticus follows the Polygonum-type and the mature female gametophyte is octonucleate (Fig. 5B–D). The embryo sacs of both A. coronaria and R. asiaticus are characterised by two typical features: (1) the presence of the large antipodal cells of which two of the three persist long after fertilization and (2) the prominent nucleus of the central cell, which is derived from the fused two polar nuclei, long before fertilization (Fig. 6A). When the fruitlets are ripe and are shed, the embryos are not yet fully developed; their growth is arrested at the torpedo stage (Fig. 6B). 3.3. Self-incompatibility Because R. asiaticus ‘Krisma’ did not produce enough pollen due to a suboptimal number of stamens, seed set could not be analysed. The remaining tested cultivars are all self-compatible based on adequate seed set except for R. asiaticus ‘Alfa’, which did not result in fruitlets, indicating self-incompatibility. To confirm the putative self-incompatibility of ‘Alfa’, in vivo pollen tube growth was tracked by aniline blue staining upon selfing. Because only a limited amount of pollen is needed for this purpose, all A. coronaria and R. asiaticus cultivars could be analysed. Of all these cultivars, only in the case of R. asiaticus ‘Alfa’ pollen tube growth in the stylar region was strongly inhibited (Fig. 7), confirming that ‘Alfa’ is largely self-incompatible. 4. Discussion In line with previous reports (Endress and Igersheim, 1999; Ren et al., 2009, 2010; Tamura, 1995c; Wang and Ren, 2008), the morphological analyses reported in this study clearly demonstrate
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the heterogeneity within the Ranunculaceae family. In the Ranunculaceae, there are four perianth types: morphologically distinct petaloid sepals and petals (e.g., Aquilegia), only petaloid tepals (e.g., Anemone), green sepals and coloured petals (e.g., Ranunculus) and green sepals and no petals (e.g., Beesia, Trautvetteria) (Kramer, 2009; Tamura, 1995c). A. coronaria has petaloid tepals, while R. asiaticus has both sepals and petals. A. coronaria has no nectaries and the studied cultivars of R. asiaticus show only rudimentary nectaries. This is in contrast with Trollius, Nigella and other Ranunculus species in which nectaries were observed which are in a pit or a pocket near the base of the petal (Tamura, 1995d). The studied cultivars of R. asiaticus showed a variable number of stamens. As both stamens and petals are arranged on the same spiral, a possible explanation is that stamens and petals can be converted in one another during tissue differentiation (Ronse Decraene and Smets, 1995; Tamura, 1995d). Indeed, early stages of petals, stamens and even carpels are hardly distinguishable. This is in line with previous studies on Anemone rivularis (Chang et al., 2005) and other studies on members of the Ranunculaceae (Ren et al., 2009). In this study, SEM confirmed the conversion of stamens to petals in R. asiaticus. Regarding gametophyte development and embryogenesis, the two genera go through similar developmental stages resulting in not fully developed embryos at the time of fruitlet shedding: the embryogenic growth is arrested at the torpedo stage. This was also observed in Helleborus niger (Niimi et al., 2006) and Hepatica nobilis (Nomizu et al., 2004), both members of the Ranunculaceae. The Ranunculus cultivar ‘Alfa’ is self-incompatible: although the pollen was able to germinate, its tube growth was arrested in the style, suggesting gametophytic self-incompatibility. This is in line with previous reports showing gametophytic self-incompatibility in some Ranunculus species (Horovitz, 1985; Lundqvist, 1990). 5. Conclusions The flower morphology of A. coronaria and R. asiaticus differs in some aspects (e.g., perianth type, nectaries). R. asiaticus showed a non conserved number of stamens, which can be explained by the conversion of stamens to petals. Seeds contain underdeveloped embryos and thus maturation is needed prior to germination. This maturation process is often accomplished by a cold treatment. For the Ranunculus cultivar ‘Alfa’ also self-incompatibility was noticed. All these characteristics are important for propagation of and breeding research using these ornamentals. Acknowledgements The authors thank T. Versluys, C. Petit and B. Meersman for technical support. Istituto Regionale per la Floricoltura and Biancheri Creations are acknowledged for supplying the rhizomes of Anemone coronaria and Ranunculus asiaticus. This work was supported by Research Foundation Flanders (FWO) to W.G. References Beruto, M., Debergh, P., 2004. Micropropagation of Ranunculus asiaticus: a review and perspectives. Plant Cell Tissue Organ Cult. 77, 221–230. Chang, H.L., Ren, Y., Lu, A.M., 2005. Floral morphogenesis of Anemone rivularis BuchHam. ex DC. var. flore-minore Maxim. (Ranunculaceae) with special emphasis on androecium developmental sequence. J. Integr. Plant Biol. 47, 257–263. Endress, P.K., Igersheim, A., 1999. Gynoecium diversity and systematics of the basal eudicots. Bot. J. Linn. Soc. 130, 305–393. Hegnauer, V.R., 1995. Vergleichende phytochemie und chemotaxonomie. In: Engler, A., Prantl, K. (Eds.), Die Natürlichen Pflanzenfamilien. Bd. 17 a IV Angiospermae. Ordnung Ranunculales. Fam. Ranunculaceae. Duncker & Humblot, Berlin, pp. 185–210. Horovitz, A., 1985. Ranunculus. In: Halevy, A.H. (Ed.), CRC Handbook of Flowering. CRC Press, Florida, pp. 155–161. Hoot, S.B., Reznicek, A., 1994. Phylogenetic relationships in Anemone (Ranunculaceae) based on morphology and chloroplast DNA. Syst. Bot. 19, 169–200.
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