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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.

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.

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.

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

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.

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).