untitled ACTA BOT. CROAT. 75 (1), 2016 39 Acta Bot. Croat. 75 (1), 39–44, 2016 CODEN: ABCRA 25 DOI: 10.1515/botcro-2016-0014 ISSN 0365-0588 eISSN 1847-8476 Embryological features, pollen and seed viability of Arnica montana (Asteraceae) – a threatened endemic species in Europe Elina Yankova-Tsvetkova*, Petka Yurukova-Grancharova, Georgi Baldjiev, Antonina Vitkova Department of Plant and Fungal Diversity and Resources, Institute of Biodiversity and Ecosystem Research, Bulgarian Academy of Sciences, 1113 Sofi a, Gagarin 2 Street, Bulgaria Abstract – The embryological features, mode of reproduction and reproductive capacity (pollen and seed via- bility) on two naturalized populations of Arnica montana in Bulgaria were studied. The embryological study shows that A. montana is a facultative apomictic species in which sexual reproduction predominates. In this species, it was established that there is a comparatively high viability of the mature pollen and embryos, which enables the successful realization of its reproductive capacity. The results of the study on A. montana reveal that both sexual and asexual vegetative reproduction with rhizomes undoubtedly play more important roles than the apomixis (namely diplospory) for support and preservation of the populations. Keywords: apomixis, Arnica montana, embryology, male and female gametophyte, pollen and seed viability, sexual reproduction * Corresponding author, e-mail: e_jankova@abv.bg; e_jankova@mail.bg Introduction Arnica montana L. is a rosette-forming perennial plant of the family Asteraceae Dum., subfamily Asteroideae, tribe Madieae (Noyes 2007). It is a diploid species with 2n = 38 (Ekenäs 2008), highly self-incompatible with ento- mophily (Luijten et al. 1996, 2000). Arnica montana has been used for medical purposes since the 1500s because of the contents of various active compounds with antiseptic, antifungal, antimicrobial and antibiotic activities. It occurs from the lowlands to the alpine belt. In the mountains, Ar- nica montana is a characteristic species of Nardus stricta grasslands and hay meadows (Oberdorfer 1994). The ex- cessive exploitation of this medicinal plant has affected the state of its natural reserves and reproductive capacity. At present A. montana is considered a rare and endemic spe- cies in Europe (Maguire 1943, Ferguson 1976, Ekenäs 2008), and is included in many European countries in the category “endangered” and listed in Annex V of the EU- FFH-directive (Directive 92/43/EEC 1992). The species is regarded as: “critically endangered” in Belgium, Bosnia, Croatia and Luxembourg; “endangered” in Belarus and the Netherlands; “vulnerable” in Estonia, Germany, Latvia, Lithuania, Portugal and Romania; and “near threatened” in Denmark and Norway. In Bulgaria, A. montana was found more than 100 years ago in the Rila Mts (Herbarium specimen SO 86331 depos- ited, exists in the Herbarium of Biological Faculty of the Sofi a University “St. Kliment Ohridski”) but up to now its occurrence in the country has not been confi rmed. Hitherto, this species has been primarily an object of chorological, karyological and phytochemical studies but scanty and fragmentary data exist on its embryology in the accessible literature. The aim of the present study is to reveal some of the main characteristics of the reproductive biology of Ar- nica montana: peculiarities of the male and female gameto- phyte; embryo- and endospermogenesis; pollen and seed viability, in connection with the realization of its reproduc- tive capacity that infl uence the shape and size of the popu- lations. Material and methods The material of two naturalized populations (two-years old) of A. montana were studied, as follows: Beli Rid ex- perimental station – on Mt Vitosha (a population from the Carpathian Mountains, Ukraine) and the experimental sta- tion in the village of Beglica – Rhodopi Mts (Western) – a population from the Botanical garden, Chemnitz, Germany. The Mt Vitosha population consists of 167 individuals and YANKOVA-TSVETKOVA E., YURUKOVA-GRANCHAROVA P., BALDJIEV G., VITKOVA A. 40 ACTA BOT. CROAT. 75 (1), 2016 the Rhodopi Mts population contains 146 individuals. Voucher specimens were deposited in the Herbarium of In- stitute of Biodiversity and Ecosystem Research, Bulgarian Academy of Sciences (SOM 168516, 168517 – from Mt Vitosha and SOM 169535 – from the Rhodopi Mts). Embryological study For embryological study, 50 fl ower buds and capitula at different stages of development were collected from 20 in- dividuals of each of the two naturalized populations (above mentioned) and fi xed in a mixture of FAA (formalin: glacial acetic acid: 70% ethanol in correlation 5:5:90 parts). Con- secutively, the plant material was treated according to the classical paraffi n methods (Sundara 2000), embedded in paraffi n and cut into 10–25 μm sections with a rotary mi- crotome. The sections were stained with Heidenhain’s hae- matoxylin and embedded in Enthelan in order to get perma- nent slides. Pollen and seed viability study Mature pollen grains were isolated from the two studied populations and their viability was estimated using the ace- tocarmine test (Singh 2003). For this purpose, anthers from 50 open fl owers from 20 plants of each population studied were collected, placed in 1% acetocarmine solution, dis- persed on the slides and the stained (viable) and unstained (unviable) pollen grains were counted in 30 anthers from each population using a light microscope (visual fi eld, en- largement 100×). For the study on seed viability, 400 mature seeds were collected from the two populations. To estimate the seed (embryo) viability, a quick tetrazolium test was applied (Pe- ters 2000). The embryos were isolated with a dissection needle, incubated in water at 30–35 °C and subsequently in a diluted 1% solution of 2,3,5-triphenyltetrazolium chloride for 24 hours. Initially, the tetrazolium solution is colourless, but it changes to red when it comes to contact with hydro- gen (a reduction process), deriving from enzymes of the respiration process of the seeds. The observations during the present study were carried out using a stereomicroscope “Leica EZ4”, LM “Olympus” CX2. The microphotographs were made with Digital Cam- era 1.4 Mpx. Results Embryological features Anther and development of the male gametophyte The anthers are tetrasporangiate. The anther wall forma- tion follows the dicotyledonous type and consists of four layers: an epidermis, an endothecium, one middle layer and a tapetum (Fig. 1A). The epidermis comprises one row of almost rectangular uninucleate cells that vastly enlarge dur- ing the anther ontogenesis. The middle layer is ephemeral and degenerates up to end of the meiosis in microspore mother cells (MMCs). The endothecium develops not clear- ly expressed fi brous thickenings of its consisting cells. This layer becomes completely disorganized at the stage of the mature three-celled pollen. Initially, the tapetum is glandu- lar, consisting of one row of uninucleate cells (Fig. 1A). During the meiosis in MMCs, a rapid lengthening and mul- tiplication of the nuclei of tapetum cells (as result of con- secutive mitoses) is observed (Fig. 1C) and they become four- to eight-nucleate at the stage of microspore tetrads. After the formation of uninucleate pollen grains, the tape- tum transforms from glandular to ameboid (Fig. 1E). At the time of anther dehiscence, the anther wall comprises only epidermis The sporogenous tissue is one-, two-rowed (Fig. 1A). The meiosis in MMCs passes with some deviations, such as: individual lagging chromosomes (Fig. 1C) and chromo- some out of the division spindle, especially during the fi rst (heterotypic) division of the meiosis. After simultaneous cytokinesis, the resultant microspore tetrads (Fig. 1B) were classifi ed on the basis of 50 enumerated tetrads, according to Schmid (1982), as: “usually” tetrahedral (71%), “occa- sionally” isobilateral (22%), “quite occasionally” T-shaped (5%) and “rarely” linear (2%). Sporadically monads and dyads were also observed. In a number of fl orets of some capitula degenerating microspore tetrads presented (Fig. 1D).At the time of shedding, the pollen grains are usually morphologically uniform, three-celled, tricolporate with Fig. 1. Anther and development of the male gametophyte: A) an- ther wall and two-rowed sporogenous tissue, B) lagging chromo- somes in the metaphase I of the meiosis in microspore mother cells, C) different type of microspore tetrads and one-rowed glan- dular tapetum with multinucleate cells, D) degenerating micro- spore tetrads in the anther locules, E) one-nucleate pollen and ameboid tapetum, F) viable and sterile pollen grains in an anther locule. Ep -epidermis, en – endothecium, ml – middle layer, tp – tapetum, st – sporogenous tissue, mt – microspore tetrad, pg – pol- len grain, atp – amoeboid tapetum, lch – lagging chromosome. Scale bars = 20 μm. EMBRYOLOGICAL FEATURES OF ARNICA MONTANA ACTA BOT. CROAT. 75 (1), 2016 41 echinate exine. Often, besides the normal viable pollen grains, small-sizedand dark-stained sterile grains were ob- served in the anthers of some fl orets in one and the same capitulum (Fig. 1F). Ovule and development of the female gametophyte The gynoecium is syncarpous, inferior with a unilocular ovary in which only one ovule forms. The well-developed ovule is anatropous, tenuinucellate and unitegmic. In it uni- cellular archesporium forms hypodermally. The archespori- um cell functions directly as a megaspore mother cell (Fig. 2A), which later undergoes meiosis to produce a linear megaspore tetrad (Fig. 2B). The embryo sac (ES) develop- ment runs according to the Polygonum (monosporic)-type from the chalazal megaspore of tetrad that functions as an embryo sac mother cell. After three mitoses, successively two-, four- and eight-nucleate ES forms. The mature ES consists of a three-celled egg apparatus (a usually pear- shaped egg cell and two synergids), two polar nuclei (after their fusion a secondary nucleus forms) and a three-celled antipodal apparatus in the chalazal part of ES (Fig. 2C). The synergids degenerate after fertilization. The antipodals are ephemeral and begin to degenerate after the formation of a secondary nucleus. The endothelium differentiates from the innermost layer of the single integument after one- nucleate ES and the cells of which it consists progressively lengthen in a radial direction during the ES development. It is important to notice that in the megaspore mother cells of some ovules in the fl orets of the capitula a restitu- tion nucleus forms after inhibited meiosis that leads to a dyad of megaspores, instead of a tetrad (Fig. 2D). Later on, in these cases, from the chalazal megaspore a meiotic diplo- sporous ES of Taraxacum-type develops. Usually, the embryo and endosperm form after double fertilization accompanied with a destruction of one syner- gid from the pollen tube penetrating it through the micro- pyle of ovule. The fi rst division of the zygote is transverse and usually runs before the endospermogenesis (Fig. 2E). The embryogenesis follows the Asterad type. In the mature seed, the embryo is nearly straight with two equal cotyle- dons. In fi ve ovules (18% from 28 observed in total with glob- ular embryo), the presence of a globular embryo together with two undamaged, long living synergids (in contrast to the stage of legitimate globular embryo in which the syner- gids are missing) provides reason to suppose that in these cases the embryo forms parthenogenetically from the unfer- tilized egg cell in ES that develops after a meiotic diplospo- rous Taraxacum-type, mentioned above (Fig. 2F). At the beginning, the endosperm passes from a free nu- cleate to a cellular stage, when a globular embryo forms. The mature seed completely lacks endosperm (Fig. 4D). Pollen and seed viability After acetocarmine staining, the cytoplasm and nuclei of viable pollen grains were stained in red while unviable, empty and shrunken pollen grains remain unstained (Figs. 3A and D). The results of the study show the high viability of the mature pollen (over 80%) in the two studied natural- ized populations of A. montana (Tab. 1). According to the intensity of staining with tetrazolium solution, the viable embryos are stained in red, while embryos partially stained Fig. 2. Ovule and development of the female gametophyte: A) megaspore mother cell in the ovule, B) lineare megaspore tetrad in the ovule, C) three-celled egg apparatus (egg cell and two syner- gides) and secondary nucleus in the embryo sac, D) megaspore dyad (initiation of the diplosporous Taraxacum-type development of embryo sac from the chalazal megaspore), E) two-celled pro- embryo and developing endosperm, F) parthenogenetic globular embryo together with preserved two synergids and nuclear endo- sperm. Mmc – megaspore mother cell, mgt – megaspore tetrad, sg – synergid, egc – egg cell, cc – central cell, md – megaspore dyad, em – embryo, sp – sperm, pem – parthenogenetic embryo. Scale bars = 20 μm (A–E) and 50 μm (F). Fig. 3. Pollen of Arnica montana analyzed by acetocarmine test: A) mature pollen grains without acetocarmine staining, B) viable pollen grains, stained in red, C) tricolporate viable pollen grain, stained in red, D) unviable, unstained pollen grain. Scale bars = 100 μm (A), and 20 μm (B–D). YANKOVA-TSVETKOVA E., YURUKOVA-GRANCHAROVA P., BALDJIEV G., VITKOVA A. 42 ACTA BOT. CROAT. 75 (1), 2016 or unstained are unviable (Figs 4A and 4D). On basis of tetrazolium testing, the seeds (embryos) were differentiated in three classes (Fig. 5): Class I – viable embryos (whole embryo stained in red); Class II – unviable embryos (only the root of embryo stained in red); Class III – unviable em- bryos (unstained). It was established that the majority of embryos are viable (68.97% 63.46% for the Mt Vitosha and Rhodopi Mts populations, respectively (Fig. 5). Discussion Embryological features Pullaiah (1983) studied the embryology of the tribe Se- necioneae, including the genus Arnica. The results of his investigation revealed only sexual reproduction in the stud- ied representatives of this tribe. In the majority of the spe- cies in the genus Arnica, which are largely polyploids, game tophytic apomixis has found – diplospory and par- thenogenetic development of the embryo (Afzelius 1936, Flovik 1940, Engell 1970). For that reason, Nordenstam (1977) excluded the genus Arnica from the tribe Senecione- ae. In the new systematical classifi cations, this genus is in- cluded in the tribe Madieae (Baldwin et al. 2002, Noyes 2007). The tapetum of the four-layered anther wall in A. mon- tana is glandular and one-layered, consisting of multinucle- ate cells. After the meiosis in the MMCs it becomes ame- boid (partial spreading of the cell walls occurs), as in most representatives of the family Asteraceae (Solntseva 1987). The formation of microspore dyads, polyads and monads in the anthers, as result of deviations during the meiosis, was often reported for apomictic representatives of the family Asteraceae (some species of the genera Crepis L., Taraxacum Cass., Rudbeckia L., Arnica L., Hieracium L. (Solntseva 1987). In contrast with the apomictic species, in which a high percentage of sterile pollen grains is ob- served (Afzelius 1936, Gustafsson 1937, Babcock and Stebbins 1938, Fagerlind 1939, Battaglia 1947), in Arnica montana we found the formation of morphologically uni- form and highly fertile pollen in the majority of the anthers. The development of the female gametophyte in A. mon- tana follows exclusively the Polygonum (monosporic)-type that begins from the chalazal megaspore of tetrad observed in the most Asteraceae (Solntseva 1987, Poddubnaya Ar- noldi 1982), namely: Chrysanthemum multicaule,(Deng at al. 2010);, Tugarinovia mongolica, (Ma and Wang 2000), Calendula offi cinalis, (Ao 2007), Chrysanthemum grandi- fl orum, (Deng et al. 2010). The Polygonum -type is not the only type for ES development in the Asteraceae family in which are observed, but more rarely, the Adoxa-type (Liu, 2001a), Drusa-type (Liu 2001b), Oenothera-type (Teng et al. 2008, Li et al. 2009) and Pyrethrum cinerariaefolium type (Hu 2005). The formation of a restitution nucleus in some ovules, a dyad of megaspores instead of tetrads as well as some other embryological peculiarities established during our study suggest that the female gametophyte de- velopment most likely follows the Taraxacum-type of mei- otic diplospory (gametophytic apomixis). Consequently, we found indications that the embryo develops parthenogeneti- cally and the proof of that is the preservation of the two synergids together with a multicellular globular embryo in the ES cavity. The same phenomenon we have observed in the species Erigeron annuus of the Asteraceae family (Yurukova-Grancharova at al. 2012) In conclusion, on the grounds that during this study in A. montana both sexual and apomictic reproduction were established, we assume that this diploid species with 2n = Тab. 1. Pollen viability in studied populations, analyzed by aceto- carmine staining. No. – number. Populations No. of pollen grains Viability (% ± SD) Germany (Rhodopi Mts) 2897 79.99 ± 4.22 Ukraine (Vitosha Mt) 2810 83.77 ± 7.12 Fig. 4. Seed (embryo) viability examined by tetrazolium test: A) seed (achene) on the right side and an isolated embryo (on the left), B) isolated viable embryo (only the root stained in red), C) unviable unstained embryo (on the left side) and viable embryo, stained in dark red (on the right), D) unviable, unstained embryo (on the left) and two viable embryos, stained in red. Scale bars = 20 μm. Fig. 5. Frequency of seeds (embryos) viability (%) assessed by tetrazolium test: 1 – Class I, viable embryos (stained in red); 2 – Class II, unviable embryos (only the root of embryo stained); 3 – Class III, unviable embryos (unstained). EMBRYOLOGICAL FEATURES OF ARNICA MONTANA ACTA BOT. CROAT. 75 (1), 2016 43 2x = 38 probably occupies an isolated position within the genus Arnica, in which polyploids with gametophytic apo- mixis (diplospory) predominate (Kao 2007, 2008; Noyes 2007). The endosperm in the family Asteraceae was shown to be usually ab initio cellular by Davis (1966). Solntseva (1987) and Poddubnaya-Arnoldi (1982) described two types of endosperm (nuclear and cellular) in this family. It was established that in Arnica montana the endosperm is not ab initio cellular, but passes a free nuclear stage. Pollen and seed viability The estimated comparatively high viability of pollen (over 80%) and seeds (68.97% 63.46% for the Mt Vitosha and Rhodopi Mts populations, respectively) of A. montana in our study provides for the successful realization of its re- productive capacity, an important condition for the preser- vation of the size of its population. Pollen viability (poten- tial fertility), seed production, viability and germination ability are closely connected to one another. In Arnica mon- tana in particular, Strykstra et al. (1998) show that lighter seeds (achenes) are better dispersed by the wind but have a lower germination ability. Conclusion In the present study the main embryological features, mode of reproduction and reproductive capacity (pollen and seed viability) in Arnica montana, a threatened endemic spe- cies in Europe, were established. The study found a high plasticity of the female generative sphere compensating for the large number of sterile ovules and degenerating ES in the fl orets of capitula observed. Besides, this high plasticity has a signifi cant importance for the better adaptation of the stud- ied species. According to the results obtained, A. montana may be considered a facultative apomictic species in which sexual reproduction predominates (only 18% of apomictic embryos were observed). 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