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In the current studies, heavy metal tolerance level, accumulation efficiency and sexual reproduction were determined in Viola × wittrockiana, a non-metallophytic ornamental cultivar in comparison to V. tricolor, a metallo- phyte, after zinc (Zn) or lead (Pb) treatment (0, 10, 100 and 1 000 ppm) in pot experiments. The seed germination frequency that was not reduced in comparison to the control, the effective Zn absorption from the soil and exclusion strategy for Pb, as well as the regular sexual reproduction of V. × wittrockiana treated with heavy metals all indicate the tolerance of this plant to heavy metals. The lack of a seed set under experimental conditions of V. × wittrockiana was due to the absence of pollinators, rather than the negative impact of heavy metals, as pollen viability and ovule development were normal under the treatments. The results indicate that V. × wittrockiana represents similar tolerance to Viola metallophytes and could be considered as a good material for the reclamation of polluted areas. The exceptional tolerance to heavy metals, the ability to initiate new generations in heavy-metal-burdened soil, which are additionally coupled with the unique beauty, make the garden pansy a good candidate to be potentially used in the future for phytoremediation purposes.

Viola banksii of the eastern Australian and Tasmanian sect. Erpetion produces exclusively chasmogamous flowers, unlike most other temperate taxa of Viola which additionally produce obligatory self-pollinated cleistogamous flowers. This study explored flower structure and nyctinastic flower movements (temporal flower closure), the correlation of the timing of nyctinastic movements with stigma receptivity, and self-compatibility of flowers. Petal movement in day/night cycles was documented by two cameras. Floral morphology, anther protuberance anatomy, stigma receptivity, and pollen tube growth were examined using scanning electron microscopy, fluorescent microscopy, histochemical and sectioning techniques. Diurnal petal movements that cause flowers to be open during the day and closed during the night were documented in V. banksii. Chasmogamous flower colour, fragrance, anterior petal venation, and the indurated green area at the base of the anterior petal are all likely to play roles for pollinator attraction. Unlike most other Viola species, the anther protuberances do not function as nectaries. The flowers offer pollinators no nectar reward. The short time of stigma receptivity (2–3 days) of individual flowers limits opportunities for insect visitation and cross-pollination. Night-closed flowers of V. banksii appear to facilitate self-pollination. Self-compatibility was confirmed by tracking the pollen tube growth from the stigma to the ovule in spontaneous and hand self-pollinated flowers. Over the floral phenological cycle, nyctinasty coincides in time with stigma receptivity and, hence, the ability of self-pollination. We discovered that the same chasmogamous flower of V. banksii can cross-pollinate while open during the day, but self-pollinate while closed during the night (i.e. acting as a cleistogamous-like flower). Several floral characters indicate that V. banksii exhibits a pollination deceit strategy. Such a pollination system has not been described elsewhere in Viola.

The special mixed reproductive system, i.e., the ability of an individual plant to develop both open, chasmogamous (CH) flowers adapted to cross-pollination and closed, cleistogamous (CL) flowers with obligate self-pollinating, is a common phenomenon in Viola L. In most sections of Northern Hemisphere violets, cleistogamy is seasonal, and CH and CL flowers develop sequentially in the season. Non-seasonal cleistogamy (simultaneous) is a rare phenomenon in rostrate violets. In the current study, we focused on modification of the CH/CL mating system in V. caspia by environmental conditions, resulting in a gradual switch from temporal cleistogamy, occurring in nature, to simultaneous cleistogamy under greenhouse conditions. V. reichenbachiana with seasonal cleistogamy was the control for V. caspia with the labile seasonal/simultaneous cleistogamy system. In simultaneous cleistogamy, the CH and CL flowers, fruits and seeds developed on an individual plant at the same time on the same branch. The typical difference between CH and CL flowers’ pistils is a straight style ending with a head-like stigma in CH and a curved style in CL adapted to self-pollination. This trait persists in the fruit and seed stages, allowing for easy recognition of fruit of CL and CH flowers in simultaneous cleistogamy. Floral meristems of CH flowers of V. reichenbachiana developed on the rhizome at the end of the growing season under short-day conditions and remained dormant until the following season. The CL floral meristems formed under long-day conditions on elongating lateral branches in the upper leaf axils. The daily temperature influenced the variable CH/CL ratio of V. caspia in nature and greenhouse conditions. Regulation of the CL/CH flower ratio by modifying environmental factors is important for basic research on genetic/epigenetic regulation of cleistogamy and for practical use to produce genetically stable lines of economically important species via CL seeds.

Abstract The paper presents a technique for micropropagation of endangered in Europe and extinct in Poland Pulsatilla vulgaris for ex situ conservation of the genetic resources. Genotype-dependent induction of somatic embryogenesis and rooting was revealed in series of two experiments (I and II) performed under the same experimental conditions. Shoot tips of seedlings were the best explants in both experiments and Murashige and Skoog (MS) medium supplemented with 0.25 or 0.5 mg L−1 BAP was suitable for induction of somatic embryos (SE) and adventitious shoots. Mass SE was obtained in experiment I after explants transfer on ½ MS (2% sucrose) + 0.45 mg L−1 B1 and extending culture to 2–3 months without passages. Rooting of adventitious shoots was a critical point. Out of seven rooting media used in experiment I, only two, ½ MS hormone free (2% sucrose) + 0.45 mg L−1 B1 or MS + 5 mg L−1 NAA + 3.76 mg L−1 B2 resulted in altogether 36.4% rooted shoots. In experiment II, somatic embryogenesis, rooting and acclimatization of adventitious shoots failed. Regenerated plantlets and seedlings converted from SE from experiment I were acclimatized to ex vitro conditions. Both genome size, determined by flow cytometry, and genetic diversity analyzed by ISSR markers, confirmed the compatibility of regenerants from experiment I with P. vulgaris initial seedlings and commercial cultivar. Regenerants obtained in experiment II differed genetically from the regenerants of experiment I and cultivar. Propagated in vitro tissues/organs (SE, adventitious shoots) of P. vulgaris could be a source of material for cryopreservation, artificial seed production and/or for acclimatization of regenerated plantlets and could be used for restoration of the extinct populations. Key Message The micropropagation technique via organogenesis and somatic embryogenesis of endangered in Europe pasqueflower was developed as a tool for species recovery. The critical point is that somatic embryogenesis is genotype-dependent, which affects the repeatability of the experiments and also imposes applying molecular techniques to confirm the genetic fidelity of the regenerants with the initial material and other genotypes.

Programmed cell death (PCD) is a process that plays a fundamental role in plant development and responses to biotic and abiotic stresses. Knowledge of plant PCD mechanisms is still very scarce and is incomparable to the large number of studies on PCD mechanisms in animals. Quick and accurate assays, e.g., the TUNEL assay, comet assay, and analysis of caspase-like enzyme activity, enable the differentiation of PCD from necrosis. Two main types of plant PCD, developmental (dPCD) regulated by internal factors, and environmental (ePCD) induced by external stimuli, are distinguished based on the differences in the expression of the conserved PCD-inducing genes. Abiotic stress factors, including heavy metals, induce necrosis or ePCD. Heavy metals induce PCD by triggering oxidative stress via reactive oxygen species (ROS) overproduction. ROS that are mainly produced by mitochondria modulate phytotoxicity mechanisms induced by heavy metals. Complex crosstalk between ROS, hormones (ethylene), nitric oxide (NO), and calcium ions evokes PCD, with proteases with caspase-like activity executing PCD in plant cells exposed to heavy metals. This pathway leads to very similar cytological hallmarks of heavy metal induced PCD to PCD induced by other abiotic factors. The forms, hallmarks, mechanisms, and genetic regulation of plant ePCD induced by abiotic stress are reviewed here in detail, with an emphasis on plant cell culture as a suitable model for PCD studies. The similarities and differences between plant and animal PCD are also discussed.

The Viola epipsila-V. palustris complex is a highly taxonomically complicated group of species in its entire circumboreal range of distribution. Habitat loss, forest flooding, and hybridization could lead to the extinction of V. epipsila. A hybrid index and principal component analysis (PCA) were used to select qualitative and quantitative morphological features to distinguish parent species and hybrids, inter simple sequence repeat (ISSR) markers to determine the genetic diversity of the populations, flow cytometry to estimate the genome size (GS), and non-coding chloroplast DNA (cpDNA) regions to indicate the directions of crosses. All taxa are very morphologically variable, and their features can change within a season. The most stable feature is the distance of the bracts on the pedicel from the rhizome. The genetic diversity of all taxa populations is low and highly influenced by selfing and vegetative propagation. The population structure is differentiated: populations of V. epipsila or V. palustris, mixed populations with both parent species, F1 hybrids and populations with introgressive forms occur in different regions. The interspecific GS variation corresponds to the ploidy level (4x = 2.52 pg, 8x = 4.26 pg, 6x = 3.42 pg). Viola epipsila is the mother plant of the hybrids. Research has shown the risk of V. epipsila extinction in Central Europe and the importance of local populations in studying the role of hybridization in reducing/maintaining/increasing biodiversity.

In vitro regeneration of Viola stagnina Kit., endangered in most part of its European distribution range, was successfully obtained based on the newly developed protocol. Adventitious shoots via direct and indirect organogenesis were induced on leaf blade and petiole fragments on solidified Murashige and Skoog (MS) medium supplemented with 0.5 or 1 mg l−1 thidiazuron, respectively. Shoots were rooted on half-strength MS medium with 2% sucrose and 0.5 mg l−1 indole-3-acetic acid (IAA), and plantlets were successfully acclimatized. Sixty-five of the regenerated plants (72% of isolated shoots cultured on rooting medium) survived at the experimental plot conditions. Among recovered via organogenesis plants, individuals ‘true-to-type’ derived from initial plant G1 and also plants genetically distant from initial plants were detected by ISSR markers. In all groups of clones genetic indices (number of genotypes, polymorphic markers, gene diversity, total gene diversity, mean gene diversity) were lower than in natural populations. Regenerated plantlets had the same genome size estimated by flow cytometry as initial material and plants from natural populations. They developed chasmogamous flowers with highly viable pollen grains (over 90%), cleistogamous flowers, and set seeds from both flower types in the first and second seasons cultivated at experimental plots. This is the first report of a successfully developed micropropagation protocol of V. stagnina, and the first detailed genetic analysis of recovered plants with the use of ISSR markers and genome size measurements allowing to discuss the advantageous role of somaclonal variation in ex situ plant conservation with the use of in vitro micropropagation.

Viola reichenbachiana (2n = 4x = 20) and V. riviniana (2n = 8x = 40) are closely related species widely distributed in Europe, often sharing the same habitat throughout their overlapping ranges. It has been suggested in numerous studies that their high intraspecific morphological variability and plasticity might have been further increased by interspecific hybridisation in contact zones, given the sympatry of the species and the incomplete sterility of their hybrid. The aims of this study were to: (i) confirm that V. reichenbachiana and V. riviniana have one 4x genome in common, and (ii) determine the impact of hybridisation and introgression on genetic variation of these two species in selected European populations. For our study, we used 31 Viola populations from four European countries, which were analysed using AFLP and sequencing of a variable plastid intergenic spacer, trnH-psbA. Our analysis revealed that V. reichenbachiana exhibited larger haplotype diversity, having three species-specific haplotypes versus one in V. riviniana. The relationships among haplotypes suggest transfer of common haplotypes into V. riviniana from both V. reichenbachiana and hypothetically the other, now extinct, parental species. AFLP analysis showed low overall genetic diversity of both species, with V. riviniana showing higher among-population diversity. None of the morphologically designated hybrid populations had additive AFLP polymorphisms that would have indicated recent hybridisation. Also, kinship coefficients between both species did not indicate gene flow. V. riviniana showed significant population subdivision and significant isolation by distance, in contrast to V. reichenbachiana. The results indicate lack of gene flow between species, high influence of selfing on genetic variability, as well as probably only localised introgression toward V. riviniana.

Programmed cell death (PCD) plays a crucial role in plant development (dPCD) and is also activated under the influence of environmental factors (ePCD). As the mechanism of PCD under heavy metal stress is poorly understood, a suspension culture of cells from the facultative metallophyte Viola tricolor was used as a model to study the role of caspase-like proteases in PCD in plant cells under heavy metal stress. Cell suspension cultures treated with Zn or Pb (2000 μM for 72 h) were subjected to TUNEL assay and ligand blotting using a biotinylated inhibitor of caspase-like proteases (biotin-xVAD-fmk). The results from the TUNEL assay showed that the frequency of PCD events was significantly higher in the metal-treated suspended cells (6.8–19.6 % vs 21.8–30.1 %, Zn or Pb, respectively) than it was in the control cells (7.1–9.1 %). The results from the biotinylated inhibitor blot analysis revealed that papain-like cysteine proteases (PLCPs) were involved in heavy metal-induced PCD. Exogenous application of the PLCPs inhibitor E-64-d at a concentration of 50 μM together with Zn or Pb remarkably increased cell viability. This is the first report on the role of the PLCPs in ePCD in plant cells under heavy metal stress.

Viola arvensis cells were selected after treatment with Zn or Pb and regenerated into plants likely to have higher tolerance levels than the initial plant. The surviving cells in the suspension treated with 2000 μM of Zn, 2000 μM of Pb or 0 μM for 72 h were maintained on a solidified half-strength MS medium supplemented with 0.5 mg L−1 TDZ to induce divisions and organogenesis. The adventitious shoots obtained were rooted on a half-strength MS medium with 1 mg L−1 IBA. Regenerants derived from the Zn- and Pb-treated cells were vigorous and fully fertile. The in vitro conditions and metal impact generated a low genome alteration and overall low genetic diversity of regenerants compared to the initial plant and plants from the natural population. The cells of regenerants obtained after Pb treatment represented an approximately 12% higher tolerance level to Pb than the cells of the initial plant. This is the first report of plant regeneration from highly tolerant cells selected by heavy metal treatment. Regenerants successfully obtained in vitro could be considered as a source material for the recultivation of areas polluted with heavy metals.

The use of cryopreservation techniques for plant species confined to wet habitats is difficult to standardize due to low tolerance for dehydration of plant tissue. The study aimed to develop a procedure for the long-term storage in liquid nitrogen (LN) of Viola stagnina, a rare and endangered species of moist habitats whose European range is constantly decreasing. Adventitious shoot apices of different sizes (small, medium and large) were cryopreserved using encapsulation/dehydration, encapsulation/vitrification (in PVS2 or PVS3 solutions) and droplet/vitrification methods. Encapsulation/dehydration was the most efficient technique, and 47.7 % regrowth was obtained with an average rate of shoot regeneration of 2.4 %. Less than 27.6 % of cryopreserved shoot apices were able to produce shoots with an efficiency of 0.35 or less based on vitrification procedures. In post-thaw recovery culture, adventitious shoots were produced indirectly (via callus). The genetic variation among recovered shoots after different cryopreservation methods was low (Dice coefficient not exceeded 0.09). Some clones were genetically uniform with the mother plant estimated by ISSR molecular markers. The genome size of all post-thaw recovered regenerants, except one individual with a multiplicated genome, was uniform with the initial plant. This paper presents the first report on cryopreservation of V. stagnina with a developed protocol that can be used in ex situ conservation of the species.

Main conclusion The distribution of cyclotides was visualized in plant cells, tissues and organs using immunohistochemistry. Finding of cyclotides in tissues potentially vulnerable to pathogen attacks supports their role as defense molecules. The cyclotide family of plant peptides is characterized by the cyclic cystine knot motif and its diverse biological activities. Given their insecticidal and antimicrobial properties, the role of cyclotides in planta is probably associated with host defense. Our current understanding of the cellular compartmentalization of cyclotides in the vacuole is based on indirect studies on transgenic model plants that do not express cyclotides naturally. Matrix-assisted laser desorption ionization (MALDI) imaging has also been used to study the distribution of cyclotides, but the technique’s resolution was insufficient to determine their tissue or cell distribution. To avoid the limitations of these approaches, immunohistochemical visualization methods were used. Antibodies were raised in rabbits using cycloviolacin O2 (cyO2), and their specificity was determined by Western and dot blot experiments. Slides for immunohistochemical analysis were prepared from leaf, petiole and root fragments of Viola odorata and Viola uliginosa, and specimens were visualized using indirect epifluorescence microscopy. The antibodies against cyclotides were specific against selected bracelet cyclotides with high similarity (cyO2, cyO3, cyO8, cyO13) and suitable for immunohistochemistry. The tissue distribution of the cyclotides visualized in this way is consistent with their proposed role in host defense—relatively large quantities were observed in the leaf and petiole epidermis in both Viola species. Cyclotides were also found in vascular tissue in all the assessed plant organs. The vacuole storage of cyclotides was directly shown. Electronic supplementary material The online version of this article (doi:10.1007/s00425-016-2562-y) contains supplementary material, which is available to authorized users.

Viola banksii, the type species of section Erpetion, is endemic in eastern mainland Australia. In this paper we characterise morphological and anatomical features and assess genome size and genetic diversity in combination with the breeding system. V. banksii develops exclusively chasmogamous flowers. Ovules are anatropous, crassinucellate and bitegmic, the female gametophyte is of the Polygonum type, and the embryo is of Asterad type surrounded by nuclear endosperm. Pollen is non-heteromorphic, 3-aperturate, and highly viable. V. banksii grows in shade on moist, well drained, often sandy soils, and this is reflected in the anatomy of its organs, which includes a lack of subepidermal collenchyma in aerial parts, large leaf epidermal cells with thin cell walls, a narrow cuticle layer, and vascular bundles with xylem that are not rich in vessels. V. banksii is tolerant to zinc and lead based on phytotoxicity test. The high chromosome number (2n = 10x = 50) does not correspond to a small genome size (2C DNA = 1.27 pg). Low mean intra-populational gene diversity (HS = 0.077) detected by ISSR markers confirms the strong influence of selfing and clonal propagation by pseudostolons. Unique morphological traits of V. banksii include nyctinastic petal movement, the lack of a floral spur, the presence of gland-like protuberances on two stamens, and the presence of pseudostolons, which could be a synapomorphy for the whole section.

Plants from Violaceae produce cyclotides, peptides characterized by a circular peptide backbone and a cystine knot. This signature motif gives stability that can harness a wide spectrum of biological activities, with implications in plant defense and with applications in medicine and biotechnology. In the current work, cyclotide expressing in vitro cultures were established from Viola uliginosa. These cultures are useful models for studying biosynthesis of cyclotides and can also be used in their production. The cyclotide expression pattern is shown to be dependent on exogenous plant growth regulators, both on peptide and gene expression levels. The highest yields of cyclotides were obtained on media containing only a cytokinin and were correlated with storage material accumulation. Exposure to auxins decreased cyclotide production and caused shifting of the biosynthesis pattern to root specific cyclotides. The response to stimuli in terms of cyclotide expression pattern appears to be developmental, and related to polar auxin transportation and the auxin/cytokinin ratio regulating tissue differentiation. By the use of whole transcriptome shotgun sequencing (WTSS) and peptidomics, 20 cyclotide sequences from V. uliginosa (including 12 new) and 12 complete precursor proteins could be identified. The most abundant cyclotides were cycloviolacin O3 (CyO3), CyO8 and CyO13. A suspension culture was obtained that grew exponentially with a doubling time of approximately 3 days. After ten days of growth, the culture provided a yield of more than 4 mg CyO13 per gram dry mass.

Zanieczyszczenia gleby metalami ciężkimi mają toksyczne działanie na rośliny, zwierzęta oraz człowieka. Metalofity, rośliny odporne na metale ciężkie, kolonizujące tereny metalonośne, są wykorzystywane do fitoremediacji, czyli oczyszczania gleb z metali ciężkich. Wykorzystanie roślinnych kultur komórkowych do badań nad toksycznością metali i tolerancją komórek na ich działanie jest stosunkowo nową techniką. W pracy zostały przedstawione możliwości wykorzystania roślinnych kultur zawiesinowych w badaniach nad wpływem metali ciężkich na metabolizm komórek oraz metody oszacowania ich toksycznego wpływu. Zaprezentowane zostały techniki otrzymywania kultur zawiesinowych, oceny żywotności komórek, akumulacji metali ciężkich w komórkach. W ocenie toksyczności metali stosuje się także badania nad programowaną śmiercią komórki (PCD), co pozwala oszacować reakcję komórek na ich wysokie stężenia. Zostały przedyskutowane mechanizmy tolerancji komórek na metale ciężkie. Kultury zawiesinowe są dobrym modelem do badań tolerancji na metale, ponieważ pozwalają zbadać ich wpływ na pojedyncze komórki w jednolitych, stałych warunkach.

We studied the zinc and lead accumulation and tolerance level of suspended cells of four Viola species with different metallophyte statuses: Viola lutea ssp. westfalica (obligate metallophyte), V. tricolor (facultative metallophyte), V. arvensis (accidental metallophyte) and V. uliginosa (nonmetallophyte), in order to determine the correlation between cell and plant tolerance. Cells of all studied species/genotypes were tolerant to metal concentrations applied to the medium for 24, 48 and 72 h, more for zinc than for lead, as estimated by cell viability using the alamarBlue assay. Viable cells of each analyzed species/genotype accumulated zinc and particularly lead in very high amounts after treatment with 2000 μM for 72 h (1500–4500 mg kg−1, 24 000–32 000 mg kg−1, respectively), determined by atomic absorption spectrometry. The bioaccumulation factor values confirmed the cells’ hyperaccumulation strategy. The cell-activated detoxification mechanism, consisting in deposition of metals in the cell wall and vacuoles, as shown by transmission electron microscopy with X-ray microanalysis, allows the cells to survive despite the high level of metal accumulation. These results indicate innate high tolerance to zinc and lead in violets with different metallophyte statuses and also in the nonmetallophyte, suggesting that evolutionarily developed hypertolerance may occurs in this group as a whole.

The development of different pollen morphs by one specimen - pollen heteromorphism - occurs in ca. one-third species of the genus Viola. Melanium section species (pansies) stand out in producing the widest range of apertures among Viola species. Aperture number decreases with elevation increase, and faster germination of five-aperturate pollen as compared with three-aperturate has previously been postulated. We re-examined pollen heteromorphism in the context of its viability, and made correlations with elevation (>1500 m a.s.l. versus

Viola uliginosa Besser is a European violet having its main distribution range in the Baltic Sea region. Today it is considered endangered and threatened. Species of Violaceae from different genera and sections are known to produce cyclotides, cyclic polypeptides of much interest due to their medicinal properties and chemical structure. The present study introduced a rare species of violet (V. uliginosa) to in vitro culture for biodiversity protection and as a model for cyclotide biosynthesis research in the Violaceae. Leaf and petiole fragments were cultured on MS medium solidified with agar and supplemented with different concentrations of plant growth regulators: TDZ, KIN and 2,4-D. Direct and indirect (via callus) organogenesis was induced on MS supplemented with TDZ (0.5 or 1 mg l−1) or with equal concentrations (2 mg l−1) of KIN and 2,4-D, followed by callus transfer on 1 mg l−1 TDZ. Shoots were rooted on MS with 2 % sucrose and 0.5 mg l−1 IBA and acclimatized. AFLP marker polymorphism was low but flow cytometry revealed that a large share of the obtained regenerants were tetraploid (2C = 4x = 2.7–2.8 pg), unlike the maternal diploid plants (2C = 2x = 1.4 pg). Eleven different cyclotides were distinguished in the aerial parts of maternal plants. Cyclotide production was significantly higher in tetraploid than in diploid plants regenerated in vitro.

Viola tricolor is a pseudometallophyte covering heavy-metal-polluted and non-polluted areas. The species is a member of the evolutionarily young sect. Melanium of Viola. In this study, we sought to determine whether the karyotype of V. tricolor is stable with respect to chromosome structure or is altered depending on environmental conditions (heavy-metal-polluted vs. non-polluted areas). We established the karyotypes of plant material originating from a Zakopane meadow (non-metallicolous population) and from the Bukowno mine waste heap (metallicolous population), showing evident interpopulation differentiation in chromosome type (2M+20 m+2sm+2st vs. 18 m+8sm), in the number, size, and distribution of rDNA loci (25S and 5S), and also in chromosome mutations, mainly fission of chromosomes into acentric fragments and translocation of the fragments. Variable numbers of both 25S and 5S rDNA loci were distributed at different positions of the chromosomes and not on specific pairs of chromosomes. The results cle...

Environmental conditions, especially pollution of soils by heavy metals, can severely affect plant morphology, anatomy and reproduction. In present investigation, the morphological variability of forest violets ( Viola reichenbachiana and V. riviniana ), their degree of colonization by arbuscular mycorrhizal fungi (AMF) and their reproduction properties were studied using samples from ore-bearing areas with soils rich in heavy metals. In these habitats, individuals with typical morphological characters of V. reichenbachiana and V. riviniana were not identified but populations were intermediate between these two species. Three ore-bearing areas with soils of a high content but low extractable amounts of heavy metals (Pb, Zn, Cd) were covered by anthropogenic pine or beech forests, contrary to typical calamine heaps with metallophyte floras. Violets growing on ore-bearing areas were fully fertile, deposited Pb and Zn mainly in roots and had low Cd levels both in shoots and roots. Plants differed in AMF colonization with the degrees varying from nil to moderate. The violets growing in these areas were not affected in their reproduction and can be considered as stabilized introgressive forms or ecotypes of parental species on these soil conditions.

The present study examines chromosome and genome size evolution in Luzula (woodrush; Juncaceae), a monocot genus with holocentric chromosomes. Detailed karyotypes and genome size estimates were obtained for seven Luzula spp., and these were combined with additional data from the literature to enable a comprehensive cytological analysis of the genus. So that the direction of karyotype and genome size changes could be determined, the cytological data were superimposed onto a phylogenetic tree based on the trnL-F and internal transcribed spacer (ITS) DNA regions. Overall, Luzula shows considerable cytological variation both in terms of chromosome number (2n = 6–66) and genome size (15-fold variation; 2C = 0.56–8.51 pg; 547.7–8322.8 Mb). In addition, there is considerable diversity in the genomic mechanisms responsible, with the range of karyotypes arising via agmatoploidy (chromosome fission), symploidy (chromosome fusion) and/or polyploidy accompanied, in some cases, by the amplification or elimination of DNA. Viewed in an evolutionary framework, no broad trend in karyotype or genome evolution was apparent across the genus; instead, different mechanisms of karyotype evolution appear to be operating in different clades. It is clear that Luzula exhibits considerable genomic flexibility and tolerance to large, genome-scale changes. © 2012 The Linnean Society of London, Botanical Journal of the Linnean Society, 2012, 170, 529–541. ADDITIONAL KEYWORDS: chromosomal evolution – chromosome numbers – chromosome size – endopolyploidy – holokinetic chromosomes – karyotype evolution – molecular phylogeny – polyploidy.

a b s t r a c t Viola tricolor (Violaceae), a species very differentiated morphologically and showing intra- and inter- population variability, occupies metalliferous (Zn, Pb, Cd, Cu) and nonmetalliferous sites through its geographic range. Here we analyzed morphological and anatomical features and also sexual reproduction in metallicolous and nonmetallicolous populations to determine whether and how they differ. Two-dimensional correspondence analysis based on selected morphological characters of vegetative and generative organs showed that plants from metalliferous soils did not form a compact group sepa- rated from those growing on nonmetalliferous soils. SEM of leaf and stipule anatomy showed differences in leaf hair ornamentation. There were significant differences in embryological processes in ovules and anthers: disturbed microsporogenesis (metallicolous 33% vs. nonmetallicolous 18%), lower pollen stain- ability (75% and 78% vs. 84% and 93%, depending on test), and higher frequency of degeneration in ovules (23% vs. 4%). These ultimately did not impede sexual reproduction of metallicolous populations but they do provide evidence that reproductive processes are sensitive to elevated heavy metals in soil and therefore can be viewed as a cost of metal tolerance. © 2011 Elsevier B.V. All rights reserved.

Changes in DNA sequences affecting cryptic intraspecific variability are very important mechanisms of plant microevolutionary processes, initiating species diversification. In polluted environments, intra- and interpopulation changes at the molecular level proceed rapidly and lead to the formation of new ecotypes in a relatively short time. We used ISSR PCR fingerprinting data to analyze the genetic diversity and genetic structure of seven populations of Viola tricolor : four growing on soil contaminated with heavy metals (Zn, Pb, Cd; waste heaps) and three from control soil. The populations from the polluted sites showed higher genetic polymorphism (% poly = 84%) and gene diversity ( H T = 0.1709) than the control populations (% poly = 75% and H T = 0.1448). The number of private markers we detected within metallicolous (MET) populations was more than double that found within non-metallicolous (NON) populations (15 vs. 7). The STRUCTURE and UPGMA analyses showed clear genetic differences between the NON and MET populations. Based on broad analyses of the genetic parameters, we conclude that the effect of these polluted environments on the genetic diversity of the MET populations, separating them from the NON populations, is evidence of microevolutionary processes at species level, leading to species divergence and the emergence of local ecotypes better adapted to their different environments.

Viola uliginosa is an endangered species throughout its range in Central Europe. Based on an unusual combination of morphological characters, including a reported lack of cleistogamous (CL) flowers, it was previously placed in the monotypic subsection Repentes, section Viola. Available individual phylogenetic analyses identify V. uliginosa as a lineage deeply embedded in section Viola, but hitherto its exact position was unknown. Here we confirm the occurrence of cleistogamy in V. uliginosa. Unlike other species of section Viola, V. uliginosa produced both chasmogamous and CL flowers at the same time, not at different times of the year (seasonal cleistogamy). The two flower types differed conspicuously in morphology and microstructure, especially in size and shape of the pistil and stamens as confirmed by scanning electron microscopy. Secondly, we resolved the phylogenetic position of V. uliginosa as a lineage deeply embedded in subsection Rostratae by means of a multigene multispecies coalescent analysis using *BEAST. In this analysis, V. mirabilis and V. uliginosa were successive sisters to the rest of subsection Rostratae and, because allopolyploids exist between V. mirabilis and the last one, subsection Rostratae needs to comprise all these taxa to be monophyletic. The evolution of key morphological traits is briefly discussed.

Two endemic zinc violets of the section Melanium Ging. occur on heavy metal soils of Central Europe. The form with yellow flowers is restricted to the area between Aachen, Germany, and Liege, Belgium, whereas the blue zinc violet exclusively thrives on a very small location at Blankenrode, Germany. Both violets are currently treated as separate species. Sequences of altogether 674 bp of the ITS1-5.8S rDNA-ITS2 regions of 61 different specimens of six taxa indicated that both violets are closely related to each other and also to Viola lutea Huds. Therefore these two zinc violets are, at best, subspecies or even only varieties of V. lutea. Thus they are termed V. lutea ssp. westfalica and V. lutea ssp. calaminaria in the present manuscript. Microsporogenesis, pollen morphology and viability of the zinc violets, particularly of the blue violet of Blankenrode, are often defective due to disturbed meiosis. The population of the blue violet might not yet be stabilized genetically but can cope with the adverse effects of the heavy metal elements.

Partial endosperm development without paternal genome involvement was induced in unpollinated ovaries of wild-type Arabidopsis thaliana cultured in vitro. Unpollinated pistils were cultured on hormone-free Murashige and Skoog (MS) medium with addition of 6% sucrose and supplemented with: benzylaminopurine (BAP; 2 mg l−1) combined with naphthylacetic acid (NAA; 0.1 mg l−1), 2,4-dichlorophenoxyacetic acid (2,4-D; explants exposed to 1-h auxin shock 20 or 40 mg l−1, and transferred to hormone-free MS medium). Initiation of autonomous endosperm (AE) development was induced on all media used in 54 ovules from 39 cultured ovaries (26%), with an average frequency of 1.4 ovules/ovary. The highest frequency of partial endosperm formation occurred on media combining the two growth regulators BAP and NAA (59% of ovaries had ovules with AE), although endosperm development was also induced on hormone-free medium (in 20.5% of ovaries). The number of AE nuclei ranged from 2 to ~50, depending on the day of culture and medium; neither cellularization nor differentiation on specific regions typical for endosperm of wild-type Arabidopsis, were noted. Fertilization independent endosperm most probably originated from the secondary nucleus, but involvement of the polar nuclei could not be excluded, as indicated by nuclear size and structure. In vitro conditions did not influence egg cell proliferation. Gynogenic embryos were observed neither in the ovules with autonomous endosperm nuclei nor in ovules without endosperm induction.

Autonomous endosperm was found in unfertilized ovules of V. odorata L. cultured on MS medium supplemented with 2,4-D as a sole growth regulator or on media with 2,4-D and BAP or kinetin. Frequency of endosperm induction was approximately 9% in ovules analyzed. The induction rate depended mainly on genotype of the donor plant, and to lesser degrees, on floral stage, flower series and medium type. Multinuclear endosperms consisting of 10–37 nuclei were found in ovules after as few as 4 days of culture. In some ovules at this stage, the egg cell and two polar nuclei were present. The process of endosperm degeneration began after 3 weeks of culture. In some ovules, degenerating autonomous endosperm was observed up to the 7th week. Parthenogenetic development of egg cells or apogamy did not accompany autonomous endosperm, supporting the hypothesis of independent pathways for embryo and endosperm development.

The aim of the study was to determine the effect of the mammalian female sex hormone estrone on differentiation of somatic tissues and on induction of autonomous endosperm in culture of female gametophyte cells of Arabidopsis thaliana ecotype Columbia (Col-0). In culture, estrone-stimulated development of autonomous endosperm (AE) occurred in 14.7% of unpollinated pistils. The AE represented development stages similar to those of young endosperm after fertilization and AE of fis mutants in vivo. In the majority of ovules the AE was in a few-nucleate young stage. Some ovules showed more advanced stages of AE development, with nuclei and cytoplasm forming characteristic nuclear cytoplasmic domains (NCDs). Sporadically, AE was divided into regions characteristic for Arabidopsis endosperm formed after fertilization. Direct organogenesis (caulogenesis, rhizogenesis), callus proliferation and formation of trichome-like structures were observed during in vitro culture of hypocotyls and cotyledons of 3-day-old seedlings cultured on medium supplemented with estrone for 28 days. Histological analysis showed adventitious root formation and changes in explant anatomy caused by estrone.

Helianthus tuberosus is economically important species. To improve characters of this energetic plant via genetic modification, production of callus tissue and plant regeneration are the first steps. A new, potentially energetic cultivar Albik was used in this study to test callus induction and regeneration. Callus was produced on leaves, petioles, apical meristems and stems from field-harvested plants but was totally non-morphogenic. Its induction started in the cortex and vascular bundles as confirmed by histological analysis. The surface of heterogeneous callus was partially covered with a membranous extracellular matrix surface network visible in scanning and transmission electron microscopies. The results clearly indicate that: (i) the morphogenic capacity of callus in topinambur is genotype dependent, (ii) cv. Albik of H. tuberosus proved recalcitrant in in vitro regeneration, and (iii) extracellular matrix surface network is not a morphogenic marker in this cultivar.

Morphological characters, AFLP markers and flow cytometry were used to investigate the morphological and genetic variability and differentiation of Viola reichenbachiana and V. riviniana in non-metallicolous (NM) and metallicolous (M) populations. The aims were to clarify the taxonomic status of plants occurring in ore-bearing areas, to determine any relationship in V. reichenbachiana and V. riviniana from sites not polluted with heavy metals, and to examine the genetic variability and differentiation of M and NM populations of both species. Multivariate analyses based on morphological characters showed significant differences between V. reichenbachiana and V. riviniana from non-polluted sites, high levels of intra- and inter-population variability, and the occurrence of inter-specific hybrids. Plants from M populations showed hybrid characters but also fell within the range of V. riviniana or V. reichenbachiana. There were no significant differences in relative genome size between plants from polluted areas and V. riviniana from NM populations. Bayesian analysis of population genetic structure based on AFLP markers distinguished two main groups: V. reichenbachiana and V. riviniana together with the M populations. That analysis also revealed the occurrence of populations of inter-specific hybrids from non-polluted areas. Further Bayesian analysis of V. riviniana including NM and M populations separated all the studied M populations from NM populations. We conclude that plants forming the M populations are well adapted to a metal-polluted environment, and could be considered as stabilised introgressive forms resulting from unidirectional (asymmetric) introgression toward V. riviniana.

In flowering plants, seeds are produced both sexually (double fertilization is required) and asexually via apomixis (meiotic reduction and egg fertilization are omitted). An apomictic-like pattern of endosperm development in planta is followed by fis mutants of sexual Arabidopsis thaliana. In our experiments in planta, autonomous endosperm (AE) developed in met1 mutants. Furthermore we obtained autonomous endosperm formation in vitro not only in unfertilized ovules of fie mutants but also in wild genotypes (Col-0, MET1/MET1, FIE/FIE) and met1 mutants. AE induction and development occurred in all genotypes on the each of the media used and in every trial. The frequency of AE was relatively high (51.2% ovaries) and genotype-dependent. AE induced in vitro represents a more advanced stage of development than AE induced in fie mutants in planta. This was manifested by a high number of nuclei surrounded by cytoplasm and organized in nuclear cytoplasmic domains (NCDs), nodule formation, division into characteristic regions, and cellularization. The high frequency of AE observed in homozygous met1 (met1/met1) mutants probably is due to accumulation of hypomethylation as an effect of the met1 mutation and the in vitro conditions. AE development was most advanced in FIE/fie mutants. We suggest that changes in the methylation of one or several genes in the DNA of Arabidopsis genotypes caused by in vitro conditions resulted in AE induction and/or further AE development.

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).

The Arabidopsis CDKG;2 gene encodes a putative cyclin-dependent Ser/Thr protein kinase of unknown biological function. This gene shows structural similarity to animal and human cyclin-dependent (PITSLRE) kinases. This study used the homozygous knockout cdkg;2 mutant based on T-DNA insertional line SALK_090262 to study the effect of mutation of the CDKG;2 gene on explant response and in vitro plant regeneration. For callus induction and proliferation, hypocotyls and cotyledons of 3-day-old seedlings of cdkg;2 and A. thaliana ecotype Col-0 were cultured on solid MS medium supplemented with 2,4-D (2 mg l-1). Organogenesis was induced after callus transfer on MS + TDZ (0.5 mg l-1). The initiation time of callus and shoot induction differed between the mutant and control cultures. Shoot regeneration after callus transfer on MS + TDZ was delayed in cdkg;2 (31 days versus 7 days in Col-0). Shoots formed on callus derived from Col-0 hypocotyls but not on cotyledon-derived callus; in cdkg;2, shoots developed on both callus types. Mutant shoots did not form roots, regenerants were dwarfed, and inflorescences had small bud-like flowers with a reduced corolla and generative organs. Abnormalities observed during cdkg;2 organogenesis suggest a role of CDKG;2 as a regulator of adventitious root initiation.

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).

Miscanthus ×giganteus Greef et Deu. (Poaceae), a hybrid of Miscanthus sinensis and M. sacchariflorus native to Japan, is an ornamental and a highly lignocellulosic bioenergy crop, cultivated in the European Union as an alternative source of energy. This grass reproduces exclusively vegetatively, by rhizomes or via expensive in vitro micropropagation. The present study was aimed at finding the barriers that prevent sexual seed production, based on detailed embryological analyses of the whole generative cycle, including microsporogenesis, pollen viability, megasporogenesis, female gametophyte development, and embryo and endosperm formation. Sterility of M. ×giganteus results from abnormal development of both male and female gametophytes. Disturbed microsporogenesis (laggard chromosomes, univalents, micronuclei) was further highlighted by low pollen staining. The frequency of stainable pollen ranged from 13.9% to 55.3% depending on the pollen staining test, and no pollen germination was observed either in vitro or in planta. The wide range of pollen sizes (25.5-47.6 μm) clearly indicated unbalanced pollen grain cytology, which evidently affected pollen germination. Only 9.7% of the ovules developed normally. No zygotes nor embryos were found in any analyzed ovules. Sexual reproduction of M. ×giganteus is severely hampered by its allotriploid (2n=3x=57) nature. Hybrid sterility, a strong postzygotic barrier, prevents sexual reproduction and, therefore, seed formation in this taxon.

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.

Violets of the sections Melanium were examined for their colonization by arbuscular mycorrhizal fungi (AMF). Heartsease (Viola tricolor) from several heavy metal soils was AMF-positive at many sites but not at extreme biomes. The zinc violets Viola lutea ssp. westfalica (blue zinc violet) and ssp. calaminaria (yellow zinc violet) were always AMF-positive on heavy metal soils as their natural habitats. As shown for the blue form, zinc violets germinate independently of AMF and can be grown in non-polluted garden soils. Thus the zinc violets are obligatorily neither mycotrophs nor metalophytes. The alpine V. lutea, likely ancestor of the zinc violets, was at best poorly colonized by AMF. As determined by atomic absorption spectrometry, the contents of Zn and Pb were lower in AMF colonized plants than in the heavy metal soils from where the samples had been taken. AMF might prevent the uptake of toxic levels of heavy metals into the plant organs. Dithizone staining indicated a differential deposition of heavy metals in tissues of heartsease. Leaf hairs were particularly rich in heavy metals, indicating that part of the excess of heavy metals is sequestered into these cells.