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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.

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.

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.

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.

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.

Cyclotides are cyclic plant polypeptides of 27-37 amino acid residues. They have been extensively studied in bioengineering and drug development contexts. However, less is known about the relevance of cyclotides for the plants producing them. The anti-insect larvae effects of kB1 and antibacterial activity of cyO2 suggest that cyclotides are a part of plant host defense. The sweet violet (Viola odorata L.) produces a wide array of cyclotides, including kB1 (kalata B1) and cyO2 (cycloviolacin O2), with distinct presumed biological roles. Here, we evaluate V. odorata cyclotides' potency against plant pathogens and their mode of action using bioassays, liposome experiments and immunogold labeling for transmission electron microscopy (TEM). We explore the link between the biological activity and distribution in plant generative, vegetative tissues and seeds, depicted by immunohistochemistry and matrix assisted laser desorption ionization mass spectrometry imaging (MALDI-MSI). Cyclotides cyO2, cyO3, cyO13, and cyO19 are shown to have potent activity against model fungal plant pathogens (Fusarium oxysporum, F. graminearum, F. culmorum, Mycosphaerella fragariae, Botrytis cinerea) and fungi isolated from violets (Colletotrichum utrechtense and Alternaria alternata), with minimal inhibitory concentrations (MICs) ranging from 0.8 to 25 mu M. Inhibition of phytopathogenic bacteria - Pseudomonas syringae pv. syringae, Dickeya dadantii and Pectobacterium atrosepticum - is also observed with MIC = 25-100 mu M. A membrane-disrupting antifungal mode of action is shown. Finding cyO2 inside the fungal spore cells in TEM images may indicate that other, intracellular targets may be involved in the mechanism of toxicity. Fungi can not break down cyclotides in the course of days. varv A (kalata S) and kB1 show little potency against pathogenic fungi when compared with the tested cycloviolacins. cyO2, cyO3, cyO19 and kB1 are differentially distributed and found in tissues vulnerable to pathogen (epidermis, rizodermis, vascular bundles, protodermis, procambium, ovary walls, outer integuments) and pest ( ground tissues of leaf and petiole) attacks, respectively, indicating a link between the cyclotides' sites of accumulation and biological role. Cyclotides emerge as a comprehensive defense system in V. odorata, in which different types of peptides have specific targets that determine their distribution in plant tissues.

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.

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 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.

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.

Chromosomes and nuclear DNA amount were analyzed in leaf tissues of Luzula nivea, Luzula luzuloides, and Luzula multiflora. Intra- and interspecific karyological variability was stated. Chromosome numbers in diploids ranged 2n = 8-24 in L. nivea and L. luzuloides and 2n = 12-84 in hexaploid L. multiflora. Karyological variability resulted mainly from chromosome fission (agmatoploidy) and aneusomaty; chromosome fusion (symploidy) and polyploidy were also involved. Flow cytometric determination of nuclear genome size using propidium iodide staining gave values of 1.584 pg in L. luzuloides, 1.566 pg in L. nivea, and 3.034 pg in L. multiflora. Variability in relative nuclear genome size within species was measured by 4',6-diamidino-2-phenylindole staining. In contrast with previous reports, variability was fairly small and ranged from 1.796 to 1.864 pg in L. luzuloides, from 1.783 to 1.847 pg and from 1.737 to 1.808 pg in two populations (S and F) of L. nivea, respectively, and from 3.125 to 3.271 pg in L. multiflora. An intraplant (interleaf) genome size variation was also observed and its possible causes are discussed.Key words: Luzula, holokinetic chromosomes, agmatoploidy, symploidy, polyploidy, nuclear DNA amount, intraplant genome size variability, flow cytometry.

Chromosome counts are reported from five species of Antarctic hepatics belonging to five genera and four families. The chromosome numbers in the genera Herzogobryum (H. teres n = 9) and Paehyglossa (P. dissitifolia n = 9) are reported for the first time. The first chromosome count from Lophozia excisa, n = 27, is the highest reported from Lophozia. A diploid number, n = 18, is reported for the first time from Barbilophozia hateheri; a second count, n = 9, agrees with earlier reports. The commonest Antarctic liverwort Cephaloziella cf. exili[lora has n = 18 and this agrees with earlier reports for the genus.

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.

Two zinc violets, the yellow form of the Aachen–Liege 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.