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Simple Summary: The wetland violets of Central and Northern Europe (Viola epipsila Ledeb., V. palustris L.) are endangered because the ranges of both species are drastically decreased due to global climatic changes. Their disappearance might also be the result of the formation of interspecific hybrids which can replace the parent species. The study of such species is particularly important because they might be considered as indicators of anthropogenic changes occurring in peatlands and their disappearances. The taxonomic situation of studied species is intricate, and the presence of interspecific hybrids and putative introgressants [V. pubifolia (Kuta) G. H. Loos (=V. palustris subsp. pubifolia Kuta)] makes it even more complicated. The main goal of our study was to reconstruct the origin of V. pubifolia and its genetic relatedness to both putative parental species—V. palustris and V. epipsila—using advanced molecular methods. The taxonomic problem has been finally solved. We found no basis to separate V. pubifolia as a species in its own right because its morphological characters as well as genetic variation fall well within the range of variability of V. palustris. We have also concluded that the low genetic differentiation and heterozygosity of V. epipsila in Europe might be a cause of the reduced tolerance of this species to changing environmental conditions and can possibly lead to its extinction. In Europe, the V. epipsila—V. palustris group comprises V. epipsila Ledeb., V. palustris L., V. pubifolia (Kuta) G. H. Loos (=V. palustris subsp. pubifolia Kuta), interspecific hybrids, and putative introgressants. The genetic affinity of V. pubifolia to V. palustris, and their shared origin via hybridization followed by polyploidization, were confirmed using inter simple sequence repeat (ISSR) markers, restriction site-associated DNA sequencing (RAD-Seq), and a low-copy nuclear gene, GPI, which encodes glucose-6-phosphate isomerase. The other taxa of subsect. Stolonosae were not identified as putative parents of V. pubifolia by GPI. Our analyses indicated that V. pubifolia can be included in the morphological and genetic variation of V. palustris. The ISSR, RAD-Seq, and genome size value separated well V. palustris from V. epipsila and hybrids. The results also reopen the discussion on intraspecific variation in the context of taxa ranks and species concepts. The reduced tolerance of V. epipsila in Europe to changing environmental conditions might result from low genetic differentiation and heterozygosity, as well as the increased number of interspecific hybrids (V. epipsila × V. palustris), and eventually can possibly lead to its extinction. The disappearance of populations/individuals of this species may indicate anthropogenic changes occurring in peatlands. [ABSTRACT FROM AUTHOR]

Arabidopsis arenosa is a good candidate for phytoremediation due to its high tolerance to Zn and Cd as well as its accumulation ability. However, its small size and low biomass are the largest obstacles to applying it on a broad scale. The aim was to obtain polyploid specimens, which tend to have higher biomass to increase the accumulation and translocation capacity of heavy metals in this metal-tolerant plant. Doubled polyploids (octaploids) were obtained via indirect organogenesis on a ½ MS medium supplemented with 1 mg L−1 TDZ, followed by rooting on the same medium without growth regulators. Callus tissue of a high endopolyploidy level (the (Ʃ>2C)/2C ratio over 2.5) obtained on seedling fragments on ½ MS supplemented with 2 mg L−1 2,4-D + 2 mg L−1 BAP served as a source material. Among the regenerants successfully obtained (without using antimitotic agents), over half, regardless of the stage of regenerant development, were octaploid (54–78%; 2C DNA = 1.642 pg). Octaploids were not affected by ploidy or in vitro culture conditions; they were fully fertile, produced normal pollen (~97% of viability), and set seeds capable of germinating (78%). Their cell and organ size was affected by genome doubling resulting in longer stomata, bigger pollen grains, and flowers with a larger area and width in comparison with tetraploid regenerants and initial plants. The promising results of measurements of morpho-anatomical, physiological, and reproductive parameters indicate that, in the future, after passing tolerance tests, the obtained polyploids could be used in phytoremediation of metal-contaminated areas. [ABSTRACT FROM AUTHOR]

The genus Viola (Violaceae) is among the 40–50 largest genera among angiosperms, yet its taxonomy has not been revised for nearly a century. In the most recent revision, by Wilhelm Becker in 1925, the then-known 400 species were distributed among 14 sections and numerous unranked groups. Here, we provide an updated, comprehensive classification of the genus, based on data from phylogeny, morphology, chromosome counts, and ploidy, and based on modern principles of monophyly. The revision is presented as an annotated global checklist of accepted species of Viola, an updated multigene phylogenetic network and an ITS phylogeny with denser taxon sampling, a brief summary of the taxonomic changes from Becker's classification and their justification, a morphological binary key to the accepted subgenera, sections and subsections, and an account of each infrageneric subdivision with justifications for delimitation and rank including a description, a list of apomorphies, molecular phylogenies where possible or relevant, a distribution map, and a list of included species. We distribute the 664 species accepted by us into 2 subgenera, 31 sections, and 20 subsections. We erect one new subgenus of Viola (subg. Neoandinium, a replacement name for the illegitimate subg. Andinium), six new sections (sect. Abyssinium, sect. Himalayum, sect. Melvio, sect. Nematocaulon, sect. Spathulidium, sect. Xanthidium), and seven new subsections (subsect. Australasiaticae, subsect. Bulbosae, subsect. Clausenianae, subsect. Cleistogamae, subsect. Dispares, subsect. Formosanae, subsect. Pseudorupestres). Evolution within the genus is discussed in light of biogeography, the fossil record, morphology, and particular traits. Viola is among very few temperate and widespread genera that originated in South America. The biggest identified knowledge gaps for Viola concern the South American taxa, for which basic knowledge from phylogeny, chromosome counts, and fossil data is virtually absent. Viola has also never been subject to comprehensive anatomical study. Studies into seed anatomy and morphology are required to understand the fossil record of the genus. [ABSTRACT FROM AUTHOR]

The study focuses on the propagation of a rare and endangered plant species (Pulsatilla patens) to re-introduce an extinct population from calamine area in Southern Poland. The plants were propagated from seeds, rhizome cuttings, or regenerated in vitro from shoot tips, hypocotyls with roots or cotyledons of seedlings on Murashige & Skoog (MS) medium supplemented with 0.25 or 0.50 mg L−1 BAP (Benzylaminopurine) via direct and indirect organogenesis or somatic embryogenesis (SE). The most efficient micropropagation method was with shoot tips as an explant on MS + 0.25 mg L−1 BAP where 97% of the explants produced multiple shoots, mass SE was observed after transfer on ½ MS with 2% saccharose; 267 (35%) shoots rooted on ½ MS + 2% saccharose were acclimatized to ex vitro conditions. Flow cytometry revealed genome size stability of propagated plantlets. Low genetic differentiation between micropropagated plantlets and initial material was indicated by ISSR (Inter Simple Sequence Repeat) markers. Totally, 132 vigorous plantlets obtained on various pathways were introduced to the field plots in 2020; 30.33% survived the winter, and several reached the generative stage and flowered in the spring 2021. In next season (March/April 2022) the number of introduced plants decreased to 25% while the number of flowering and fruiting shoots in different clumps increased in some plots. This is the first report of successful re-introduction of the endangered P. patens based on micropropagation, rhizome cuttings, and seed germination. [ABSTRACT FROM AUTHOR]

Cyclotides are macrocycle peptides produced by plants from several families, including Violaceae. These compounds have the potential for applications in medicine, bioengineering and crop protection thanks to their multiple biological activities. In most cases, cyclotides are extracted from plant material. Plant cell culture provides a viable and sustainable form of plant biomass production Cyclotides are host defense peptides. The aim of the current study was to test whether different plant stress hormones and biological elicitors have effects on cyclotide production in Viola uliginosa suspension cultures. Different concentrations of jasmonic acid (JA), salicylic acid (SA), abscisic acid (ABA) and neutralized pathogens were tested. The cyclotide production was assessed using MALDI-MS. Five major peptides produced by V. uliginosa cultures were chosen for analysis, of which one was sequenced de novo. The treatments had little influence on the suspension's growth, with the exception of 100 μM SA, which enhanced the biomass increase, and 100 μM ABA, which was toxic. Significant increases in the production of three cyclotides (viul M, cyO13 and cyO3) were observed in suspensions primed with JA (50 μM, 100 μM, 200 μM) after 14 days of culturing. Biotic elicitors had no observable effect on cyclotide production. The current study indicates that some cyclotides in V. uliginosa are triggered in response to JA. The stress plant hormones can be used to enhance plant cell culture-based production systems. [ABSTRACT FROM AUTHOR]

Plants employ different chemicals to protect themselves from herbivory. These defenses may be constitutive or triggered by stress. The chemicals can be toxic, act as repellents, phagosuppressants and/or phago-deterrents. The two-spotted spider mite (Tetranychus urticae) is a generalist arthropod herbivorous pest and its feeding causes extensive damage both to crops and wild plants. Cyclotides are cyclic peptides involved in host-plant defenses. A single Viola sp. can produce more than a hundred cyclotides with different biological activities and roles. The organ and tissue specific cyclotide patterns change over the seasons and/or with environment, but the role of biotic/abiotic stress in shaping them remains unclear. Here, we demonstrate the involvement of cyclotides in mutual interactions between violets and mites. We used immunohistochemistry and mass spectrometry imaging to show the ingested cyclotides in T. urticae and assess the Viola odorata response to mite feeding. Moreover, to assess how mites are affected by feeding on violets, acceptance and reproductive performance was compared between Viola uliginosa, V. odorata and Phaseolus vulgaris. We demonstrate that cyclotides had been taken in by mites feeding on the violets. The ingested peptides were found in contact with epithelial cells of the mite digestive system, in the fecal matter, feces, ovary and eggs. Mites preferred common bean plants (P. vulgaris) to any of the violet species; the latter affected their reproductive performance. The production of particular cyclotides in V. odorata (denoted by molecular weights: 2979, 3001, 3017, 3068, 3084, 3123) was activated by mite feeding and their levels were significantly elevated compared to the control after 5 and 21 days of infestation. Specific cyclotides may affect mites by being indigestible or through direct interaction with cells in the mite digestive tract and reproductive organs. A group of particular peptides in V. odorata appears to be involved in defense response against herbivores. [ABSTRACT FROM AUTHOR]

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 metallophyte, 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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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 (2 n = 10 x = 50) does not correspond to a small genome size (2C DNA = 1.27 pg). Low mean intra-populational gene diversity (H S = 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. Viola banksii occurs exclusively in Australian flora and is a type species of the native, Australian Erpetion section of the genus Viola. Some traits described in these studies are new for the Viola genus and could be unique for the Erpetion section. The Australian flora is particularly interesting for the study of evolution because of its long isolation from other continental floras, the high degree of endemism, and its distinctive character. [ABSTRACT FROM AUTHOR]

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 μM to 25 μM. Inhibition of phytopathogenic bacteria – Pseudomonas syringae pv. syringae, Dickeya dadantii and Pectobacterium atrosepticum – is also observed with MIC = 25–100 μM. 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. [ABSTRACT FROM AUTHOR]

Abstract: 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 <1500 m a.s.l.), soil type (metalliferous versus non‐metalliferous; MET versus NMET) and chromosome number based on selected study criteria of ca. 20% karyologically and morphologically strongly differentiated but genetically closely related pansies. A total of 79% of analysed species were heteromorphic, forming three‐ to six‐colp or ate pollen per individual flower. Mean aperture number and pollen viability were not affected by soil type (MET versus NMET). Mean aperture number was also not influenced by elevation or species chromosome number. Positive correlations were established between aperture number and pollen viability, negative between pollen viability and elevation (increasing altitude of 100 m decreased pollen viability by 0.4%) and lack of correlation between chromosome number and pollen viability. The varied frequencies of different pollen morphs among species are not under the general pressure of ecological conditions, as previously postulated for the species of Melanium section. Rather, this trait in pansies, similar to other floral characters (e.g. long, curved nectar spur, ‘landing platform’, posterior petals with nectar guides), is adaptive but dependent on the breeding system (inbreeding versus outbreeding) of the individual species. [ABSTRACT FROM AUTHOR]

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The article describes the usefull tools to study regeneration processes in plant tissue culture. It attempts to choose the most convenient method to obtain regenerated Viola plants with maternal genotype, via direct organogenesis or somatic embryogenesis. It examines the effect of mutation on CDKG.

Viola uliginosa (bog violet) is a declining species throughout its range due to - mostly anthropogenic - drying out of the wet habitats it occupies. Using AFLP markers, we aimed to estimate the genetic diversity in Polish populations, that may give an insight into the situation of plant populations facing rapid loss of natural habitats. Bog violet from several dispersed Polish populations is generally characterized by very low genetic diversity ( HT = 0.048), even lower than several other endangered violets; therefore, we suggest that it should preserve at least EN rank in the red lists/red data books. The mean gene diversity within all populations ( HS) was much lower than gene diversity ( GST) between populations (0.020 versus 0.583, respectively) which supports the prevalence of clonal propagation of the species (mainly by stolons) but may also point to some significance of autogamy in cleisto- and chasmogamous flowers. A high FST value and the Mantel test for all populations revealed significant isolation by distance. Geographically neighboring pairs of populations formed genetic clusters supported by all (in the case of two closest populations) or most statistical analyses applied. Special attention should be paid to the locus classicus of the species in Rząska, consisting of a small number of individuals, forming a genetically distinct group, revealing very low gene diversity ( Hj = 0.009) and the longest genetic distance to the remaining populations. Our results can contribute to planning future protection measures for the species at this and other locations. Genetic structure of the studied populations suggests local affinities of populations but does not generally support hypothesized recent continuity of V. uliginosa range along the river valleys of southern Poland; this view may, however, be altered with widening of the scope of studied populations and chosen molecular markers. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

The article discusses the usefulness of morphological characters in determination of intra- and interspecific diversity of violets or violaceae. It analyzes the intra- and interpopulation differentiation in Viola tricolor L. To determine the differences influenced by environmental conditions, the morphological and anatomical features in metallicolous and non-metallicolous populations are examined.

The author informs us about him taking over as the new Editor-in-Chief for Acta Biologica Cracoviensia Series Botanica, replacing Professor Elzbieta Kuta. He expresses his commitment to keep up with the good work done by her and conveys the gratitude of the entire staff, for her dedicated service. He also states his future plans to improve the quality of the journal and requests the support of his team and authors, inviting them to publish in the journal.

The article discusses the investigation of areas polluted with heavy metals acting as laboratory for microevolutionary & speciation processes of Violaceae family .

The article discusses the characterization of floral structure and pollen morphology in taxonomy of the genus Viola or violaceae. Using the scanning electron microscopy (SEM), the microstructural characters of generative organs and pollen in representatives of three sections were analyzed in details, It suggests that the micromorphological characters of flowers are also useful in reconstruction of closely related species origin.