ACTA BIOLOGICA CRACOVIENSIA Series Botanica 57/2: 89–97, 2015 DOI: 10.1515/abcsb-2015-0018 HISTOLOGICAL AND CYTOLOGICAL ANALYSIS OF MICROSPOROGENESIS AND MICROGAMETOGENESIS OF THE INVASIVE SPECIES GALINSOGA QUADRIRADIATA RUIZ & PAV. (ASTERACEAE) JOLANTA KOLCZYK, MONIKA TULEJA* AND BARTOSZ JAN PŁACHNO Department of Plant Cytology and Embryology, Jagiellonian University in Kraków, Gronostajowa 9, 30-387 Kraków, Poland Received April 27, 2015; revision accepted May 20, 2015 Galinsoga quadriradiata Ruiz & Pav. is an annual weedy plant that can be found all over the world. It belongs to the Asteraceae family and is recognised as one of the invasive foreign plants in Poland, which are native to Central and South America. The aim of this study was to describe the reproductive features of Galinsoga quadri- radiata focusing on the changes that occur during microsporogenesis and microgametogenesis along with the morphology of its pollen. As it is typical of the eudicot clade of Angiosperms, cytokinesis of G. quadriradiata is simultaneous. The pollen grains are tricolporate with spiny outer walls and the course of the microsporogenet- ic process is fairly typical of the Echinatae group of weed plants. The high viability of the pollen grains, which mature unequally in the inflorescences, and the proper course of meiosis determine whether a plant has the invasive character of Galinsoga quadriradiata. Key words: G. quadriradiata, microgametogenesis, microsporogenesis, pollen grains, invasive kenophyte, weed plant INTRODUCTION Seeds are able to germinate immediately upon con- tact with warm and moist soil, and therefore each The main process in Angiospermae that leads to growing season the plant can achieve two to three production of pollen, which represents a male game- generations (Reinhardt et al., 2003). Seeds are dis- tophyte, is meiosis. This process containing a series persed mainly by the wind or by animals (Reinhardt of tightly controlled events, which occur in the et al., 2003). anthers, can be divided into two major processes – G. quadriradiata is a weedy and invasive plant, microsporogenesis and microgametogenesis which causes economic damage to crop cultures as (Bedinger, 1992). Microsporogenesis starts with the it is a strong competitor in weedy plant communi- beginning of the meiosis of the diploid mother cells ties. It takes up nutrients that are necessary for the and ends with the formation of haploid growth of cultivated plants and because of its rela- microspores. When the microspores are released tively large leaf surface area it may shade out the cul- from the tetrads, microgametogenesis starts and tivated plants (Reinhardt et al., 2003). Moreover, after subsequent divisions, complete male gameto- according to Huffman (2004), Galinsoga species phytes are formed. serve as alternate hosts for many insects, viruses Our studies concern the events and changes and nematodes that affect crop species. that occur during microsporogenesis and microga- The genus Galinsoga Ruiz & Pav. comprises 14 metogenesis that lead to formation of mature pollen species (Cannie, 1977), which are mainly confined grains in Galinsoga quadriradiata Ruiz & Pav. This to Mexico as well as Central and South America, species is an annual plant that flowers from June to except for G. parviflora Cav. and G. quadriradiata late autumn. An eight- to nine-week-old plant can (Gopinathan and Babu, 1982). The native range of produce 3000 flower heads and a large number of G. quadriradiata covers parts of South and Central seeds, up to 7500 (Kagima, 2000; Huffman, 2004). America from Mexico to Chile, but due to human *e-mail: [email protected] PL ISSN 0001-5296 © Polish Academy of Sciences and Jagiellonian University, Cracow 2015 90 Kolczyk et al. activity the species has spread far from its original dine blue O (TBO) and mounted in Enthellan syn- range (Kabuce and Priede, 2010). In Poland, the thetic resin (Merck). species was first found near Wrocław in the second In order to detect insoluble polysaccharides, the half of the 20th century, while the first herbarium sections were oxidised for 30 min in 1% periodic records in Poland date back to 1876 (Tokarska- acid, rinsed in distilled water for 10 min, stained in Guzik, 2005). It is now a common crop weed and Schiff's reagent for 30 min, washed twice in 0.5% ruderal plant here. G. quadriradiata is often found potassium metabisulfite for 10 min each and rinsed on arable lands, roadsides, railways and in gardens under running water for 45 min. where it is highly competitive and spreads quickly For histological observations, the flower heads often becoming the dominant species in a field were also fixed in 96% ethanol and glacial acetic acid (Kabuce and Priede, 2010). It is adapted to a warm (v/v 1:3) and then stored in 70% alcohol at 4°C. After climate and heavy, nitrogen-rich soils (Anonymous, dehydration in an ethanol series and embedding in 1996). paraffin, the material was sectioned on a microtome Because asexual reproduction has been detect- (10 μm thick), stained with Heidenhain's hema- ed in Asteraceae species (and connected with some toxylin with alcian blue and mounted in Enthellan abnormalities in pollen development), we wanted to synthetic resin (Merck). check microsporogenesis and pollen morphology in The viability of the pollen grains was tested G. quadriradiata Ruiz & Pav., especially since using the acetocarmine test and Alexander test. For Galinsoga species were suspected in the case of both methods, young fresh inflorescences of apomixis (Pietrusiewicz et al., 2005). This seems to G. quadriradiata were fixed in 96% ethanol and gla- be valuable because of the interesting nature of this cial acetic acid (v/v 1:3). In acetocarmine (1% aceto- plant and the adaptations that support its invasive carmine) staining, the cytoplasm of viable pollen properties. This description of the development of grains stains red while it remains transparent in the male gametophyte, cytological aspects and non-viable pollen grains. Alexander's dye-stuff (a pollen morphology can constitute a compendium of mixture of malachite green that stains the cellulose the embryological events of this weedy and invasive of the pollen wall green and acid fuchsin that dyes plant together with female embryological studies the pollen grains protoplasts red) shows viable (Kolczyk et al., 2014). Moreover, to the best of our pollen grains that are red while nonviable pollen knowledge, there is lack of such studies in the liter- grains stain green (Alexander, 1969). ature and the embryological aspects are very impor- The Technovit microscopy and paraffin sections tant since Galinsoga quadriradiata propagates only were examined using a Nikon Eclipse 400 light generatively (Jursik et al., 2003). microscope and photographed with a Zeiss Axio Cam MRe digital camera. MATERIALS AND METHODS RESULTS The flower buds and flower heads of Galinsoga quadriradiata (Fig.1a) at different developmental THE MICROSPORANGIUM WALL stages were collected from plants in their natural habitats (wastelands, roadsides) in Kraków. For The flower of G. quadriradiata possessed five sta- light microscopy, the plant material (the flower mens. Each stamen had four microsporangia that buds) were fixed in 5% buffered (0.1 M phosphate were arranged in pairs in the two symmetrical lobes buffer, pH 7.2) glutaraldehyd for three hours at (Fig. 1b), which were joined by a connective tissue room temperature, washed four times in a phos- with a centrally located vascular band. The anthers phate buffer and then dehydrated in a graded were elongated in the longitudinal sections (Fig. 1c) ethanol series: 10%, 30%, 50%, 70%, 96%, 15 min and each microsporangium was surrounded by a each. Then the plant material was kept overnight in wall that consisted of four visible layers of the cells absolute ethanol and subsequently the samples were as follows: the epidermis as the external layer, a infiltrated for 1h each in 3:1, 1:1 and 1:3 (v/v) slightly deeper cell layer – the endothecium, a mid- mixture of absolute alcohol and Technovit 7100 dle layer and the innermost tapetum (Fig.1b). Only (2-hydroxyethyl-methacrylate) (Heraeus Kulzer). anticlinal divisions were observed among the epi- The samples were embedded in pure Technovit for dermal cells as well as within the endothecium cells. 12 hours, followed by polymerisation of resin with The middle layer mostly consisting of parenchyma addition of hardener. The plant material was sec- cells, was very fragile and disappeared very quickly tioned to 7 μm on a rotary microtome (Microm, during pollen development and was no longer visible Adams Instrumenten). The sections were floated on at the stage of young tetrads. The tapetum cells were water on a clean slide and dried to settle the sections much larger than the cells of the other layers of the onto a slide, then they were stained with 0.1% tolui- pollen sac wall. The histological analysis revealed Microsporogenesis of Galinsoga quadriradiata 91 Fig. 1. General outline of inflorescence and several stages of pollen development of Galinsoga quadriradiata Ruiz&Pav. (a) A view of an inflorescence with compact arrangement of individual flowers with jutting stigmas of the pistils. Bar: 0.5 cm. (b) A sample of transversal section of pollen sacs with visible archesporial tissue and microspore mother cells (MMCs) marked with arrow. (Toluidine blue staining). Bar: 20 μm. (c) A singular flower section with clear- ly viewed 2 anthers containing microspores (PAS reaction). Bar: 50 μm. (d) A longitudinal section of an anther with good visible multinucleated tapetum cells, tetrads and the presence of cytoplasmic channels between them (arrows). (Toluidine blue staining). Bar: 20 μm. (e) Two microspores with specific spiny apertured walls visible on the cross section of a singular anther and peryplasmodium among them. (Toluidine blue staining). Bar: 20 μm. (f) A cross sec- tion of a pollen sac with visible callose layer around the tetrads (arrows). (Toluidine blue staining). Bar: 20 μm.