Floral structure and pollen morphology of two zinc violets (Viola lutea ssp. calaminaria and V. lutea ssp. westfalica) indicate their taxonomic affinity to Viola lutea

Two zinc violets, the yellow form of the Aachen–Liège area and the blue morph of Blankenrode in western Westphalia, have very restricted occurrence on heavy metal waste heaps. Their taxonomic affinities have not been finally resolved. The flower micromorphological analysis presented here indicates that both zinc violets are closely related to the alpine Viola lutea, in line with our earlier published molecular data, but not with the conclusions of other authors. The zinc violets are classed at the rank of subspecies as V. lutea: ssp. calaminaria for the yellow zinc violet and ssp. westfalica for its blue counterpart. Although the violets examined (V. lutea, V. lutea ssp. calaminaria, V. lutea ssp. westfalica) are closely related, there is no evidence that V. lutea ssp. westfalica is a descendent of V. tricolor. Here we provide the most detailed information on generative organ structure in the four violets studied

Influence of a heavy-metal-polluted environment on Viola tricolor genome size and chromosome number

Intraspecific changes in genome size and chromosome number lead to divergence and species evolution. Heavy metals disturb the cell cycle and cause mutations. Areas contaminated by heavy metals (metalliferous sites) are places where microevolutionary processes accelerate; very often only a few generations are enough for a new genotype to arise. This study, which continues our long-term research on Viola tricolor (Violaceae), a species occurring on both metalliferous (Zn, Pb, Cd, Cu) and non-metalliferous soils in Western and Central Europe, is aimed at determining the influence of environments polluted with heavy metals on genome size and karyological variability. The genome size of V. tricolor ranged from 3.801 to 4.203 pg, but the differences between metallicolous and non-metallicolous populations were not statistically significant. Altered chromosome numbers were significantly more frequent in material from the polluted sites than from the non-polluted sites (43% versus 28%). Besides the standard chromosome number (2n = 26), aneuploid cells with lower (2n = 18–25) or higher (2n = 27, 28) chromosome numbers were found in plants from both types of site, but polyploid (2n = 42) cells were observed only in plants from the metalliferous locality. The lack of correlation between chromosome variability in root meristematic cells and genome size estimated from peduncle cells can be attributed to elimination of somatic mutations in generative meristem, producing chromosome-stable non-meristematic tissues in the peduncle

Critical factors besides treatment dose and duration need to be controlled in Pb toxicity tests in plant cell suspension cultures

The study was designed to determine the proper conditions for suspension culture of Viola tricolor cells in toxicity studies of Pb at different concentrations (0, 200, 500, 1000, 2000 \mu M) and exposure times (24, 48, 72 h). By forming insoluble salts with ions from the medium, lead (II) nitrate added to the medium decreased the initial 5.7-5.8 pH of the medium, depending on the Pb salt concentration and light intensity. In alamarBlue assays, we found no dose- or time-dependent effect of Pb on cell viability when we did not adjust pH and did not standardize the illumination conditions to correct the effect of lead-salt-induced turbidity. When effective illumination was adjusted to correct for turbidity at the highest lead concentration and pH was adjusted to 5.7-5.8, cell viability decreased with the increase of Pb(NO_{3})_{2} concentration and with treatment time. These experiments demonstrate that the toxic action of lead on cells in suspension depends strongly on culture conditions, and not only on the metal concentration and duration of treatment

Floral structure and pollen morphology are important characters in taxonomy of the genus Viola (Violaceae)

the pistil with stigma, stamen appendages (nectaries) and pollen heteromorphism are important diagnostic features in the genus ViolaL. The style characters were crucial in the very early classifications of this genus (Clausen 1927). We analyzed in details, using scanning electron microscopy (SEM), the microstructural characters of generative organs (style and stigma, stamens with nectaries) and pollen in representatives of three sections (Viola L., Melanium Ging., DischidiumGing.) occurring in Poland to get insights into the relatedness among far-related (different sections) and closely related (sub-sections within section) species. There is a great difference in stigma micromorphology between sections. In the section Violaflowers have style beaked at the apex, glabrous or covered by papillae and/or hairs, depending of subsection. Monotypic section Dischidiumwith one species V. biflora L. characterizes 2-lobed stigma. Cup-shaped stigma with the hole on the top and a lip below, covered with papillae and hairs on its outer surface occurs in pansies of the section Melanium. Pollen is highly heteromorphic (different pollen morphs, from three up to six apertures within one flower or even within one pollen sac) in the Melaniumsection and weakly heteromorphic mainly with three apertures in diploids of Viola and Dischidiumsections. This character is independent of the polyploidy in the Melaniumbut not in Violasection (Dajoz 1999). The flower micromorphological characters are also useful in reconstruction of closely related species origin. Based on stigma and nectaries features, two zinc violets are more similar to the alpineV. lutea, than to V. tricolor, indicated also as the ancestor (Kuta et al.2012)

Usefulness of morphological characters in determination of intra- and interspecific diversity of violets (Viola L., Violaceae)

The genus ViolaL. with more than 500 species is difficult taxonomic group due to great intraspecific morphological variability resulted from the influence of environmental conditions but also from hybridization. Interspecific hybrids form hybrid swarm or introgressants, therefore species discrimination still remains a problem in this genus. To analyze intra- and interspecific variability the species from two sections were studied. Intraspecific (intra- and interpopulation) differentiation were analyzed in V. tricolorL. (section Melanium) from non-polluted and polluted with heavy metals sites. Intra- and interspecific variability with the influence of hybridization were studied in V. reichenbachiana Jord. ex Boreau – V. riviniana Rchb. group (section Viola). V. tricoloris a species very differentiated morphologically (intra- and interpopulation variability). It occupies metalliferous (Zn, Pb, Cd, Cu) and non-metalliferous sites in its geographic range. We analyzed morphological and anatomical features in metallicolous and non-metallicolous populations to determine the differences influenced by environmental conditions. In field observations, plants within both population types were quite variable, but interpopulation differences in some characters were evident (plant height, stem branching, number of flowers per plant, hairiness of whole plant and leaf, leaf shape and color, petal color, plant height, spur length, seed length). Correspondence analysis (CA) based on a data matrix of 12 selected qualitative and quantitative characters indicated that in terms of morphological variation the metallicolous populations did not possess common characters separating them from the non-metallicolous populations (Słomka et al. 2012). V. reichenbachianaand V. rivinianaare closely related species and due to a lack of prezygotic isolation mechanisms they hybridize in nature and easily produce interspecific hybrids and introgressive forms which occur sympatrically. Multivariate statistical analyses (MSA) based on 50 morphological characters of vegetative and generative organs showed significant differences between V. reichenbachianaand V. riviniana from non-polluted by heavy metals areas, high level of its intra- and interpopulational variability and the occurrence of interspecific hybrids. Plants from metal-polluted sites were very viable and formed a cluster of intermediate morphotypes between V. reichenbachianaand V. riviniana with some individuals included into V. riviniana range (Migdałek et al.2013, in press)

Reduced pollen viability and achene development in Solidago ×niederederi Khek from Poland

Pollen and achene characters of natural interspecific hybrid Solidago ×niederederi Khek were analyzed and compared with putative parental species S. virgaurea L. and S. canadensis L. to estimate the level of disturbances in generative reproduction resulting from its hybrid nature. Pollen viability (stainability) of Solidago ×niederederi from one newly discovered locality in NE Poland was evidently reduced to ~65% in both viability tests (acetocarmine and Alexander). The diameter of viable pollen (median 21.11 µm) fell between S. canadensis (median 19.52 µm) and S. virgaurea (median 23.48 µm). Both parental species produced normally developed achenes with high frequency (~90%) whereas in the hybrid, the seed set was dramatically low (6%). The results clearly indicated that sexual reproduction of hybridogenous taxon S. ×niederederi is disturbed, and its potential impact as an invasive species depends mainly on vegetative propagation

Failure of androgenesis in Miscanthus × giganteus in vitro culture of cytologically unbalanced microspores

Miscanthus × giganteus is a popular energy crop, which due to its hybrid origin is only vegetatively reproduced. Asexual embryogenesis in anther and microspore culture leading to double haploids production could allow to regain the ability for sexual reproduction and to increase the biodiversity of the species. Therefore, the goal of this paper was to investigate the requirements of androgenesis in Miscanthus. The standard protocols used for monocotyledonous plants were applied with many modifications regarding the developmental stage of the explants at the time of culture initiation, stress treatment applied to panicles and isolated anthers as well as various chemical and physical parameters of in vitro culture conditions. Our results indicated that the induction of androgenesis in M. × giganteus is possible. However, the very low efficiency of the process and the lack of regeneration ability of the androgenic structures presently prevent the use of this technique

Přirozená hybridizace mezi Gladiolus palustris and G. imbricatus, zjištěná na základě morfologických, molekulárních a reprodukčních znaků

While studying the extremely rare species, Gladiolus palustris, in Poland, putative hybrid plants were discovered. Natural hybridization between G. palustris and G. imbricatus was confirmed by chloroplast (psbA-trnH and rpl32-trnL) DNA and nuclear ribosomal DNA (ITS1) sequences AFLP markers and macro-, micromorphological and reproductive characters. Based on molecular data, the hybridization events are likely to have occurred relatively recently with G. palustris as the maternal species and G. imbricatus as the pollen donor in interspecific crosses. The existence of a shared common cpDNA haplotype in all hybrids and G. palustris indicates unidirectional hybridization. A new nothospecies, G. ×sulistrovicus, is described. Analyses of AFLP data and polymorphisms of ITS1 sequences showed additive inheritance of parental genomic fragments in G. ×sulistrovicus. The hybrids exhibited either morphological similarity to G. imbricatus or intermediateness in phenotypic characters. The corm structure of flowering plants and seed capsules clearly distinguish the hybrid. The new taxon is characterized by a lower generative reproduction than the parental species, however hybrids produce ~50% viable pollen and seeds, which allows them to produce subsequent hybrid generations. The weak generative reproduction was enhanced by highly efficient vegetative propagation. The western part of the Balkan Peninsula and adjacent areas (Croatia, Bosnia and Hercegovina, Serbia, northern Italy) and central Europe (Poland, the Czech Republic, Slovakia, eastern Austria, Hungary) are the most likely areas where G. ×sulistrovicus will occur. Hybridity in the context of G. palustris conservation is discussed