Comprehensive Embryological Analyses of Common Buckwheat (Fagopyrym esculentum Moench).

Knowledge of detailed reproductive biology of cultivated species is important as requirements for fruit and seed production allow the development of effective management strategies and a sustainable use. Embryological processes of common buckwheat (Fagopyrum esculentum Moench) are difficult to interpret due to the influence of genetic determinants, i.e., dimorphic heterostyly resulting in the production of long- and short-styled flowers, and environmental predisposition, i.e., sensitivity of ovules to thermal stress. Furthermore, the situation is complicated by overproduction of flowers and depletion of resources as the plant ages. Herein we provide protocols that allow to visualize both basic and more specific embryological features and also disturbances in sexual reproduction of common buckwheat resulting from external and internal factors. All stages of plant material fixation, preparation, staining, and observation are described and explained in detail. Technical tips and pictures of properly prepared microscopic sections are also provided.

The yield of common buckwheat (Fagopyrum esculentum Moench) depends on the genotype but not on the Pin-to-Thrum ratio.

Common buckwheat has a complicated flowering biology. It is characterized by a strong self-incompatibility resulting from heterostyly, i.e. the occurrence of two types of flowers: Pin and Thrum, differing in the length of pistils and stamens. Fertilization occurs only as a result of cross-pollination between these morphs. Suspicions exist that the disturbed ratio between plants producing Pin and Thrum flowers (with the latter type generating more seeds) causes low seed yield. The aim of the study was to analyze: (1) the ratio between plants with Pin and Thrum morphs, (2) flower and seed production, as well as abortion of flowers, (3) the composition of nectar collected at an early flowering stage and during full flowering. The study was performed under semi-controlled and field conditions on six Polish accessions. The results indicated that under semi-controlled conditions the Pin-to-Thrum ratio was indeed disproportionate; such a phenomenon is called anisoplethy. In the field, however, the Pin-to-Thrum ratio was well-balanced (isoplethy). The plants with both morphs aborted a similar percentage of flowers and produced a comparable number of empty seeds. The number of flowers, their abortion, and ripe seed production were independent of flower type, however, they were genotypically controlled. A strong correlation between the number of flowers produced by a plant, flower abortion and the number of empty seeds was found. The percentage of aborted flowers correlated positively with the weight of ripe seeds. Nectar composition was similar for all buckwheat genotypes, but we found some differences in the amount of individual sugars depending on the blooming stage. In the majority of accessions, the nectar produced at the early blooming stage was characterized by a greater mass and volume, and contained more individual sugars than at the full-flowering stage.

The Effect of Stimulants on Nectar Composition, Flowering, and Seed Yield of Common Buckwheat (Fagopyrum esculentum Moench).

Common buckwheat is a valuable plant producing seeds containing a number of health-promoting compounds and elements. Buckwheat does not contain gluten and is characterized by an excellent composition of amino acids. This species is also a melliferous plant. Despite many advantages, the area of buckwheat cultivation is decreasing due to unstable yields. One of the reasons for low seed yield is its sensitivity to drought, high temperatures, and assimilate deficiencies. These factors have a significant impact on the nectar composition, which is important for visiting pollinators and thus for pollination. High temperature during flowering increases the degeneration of embryo sacs and embryos, which is high anyway (genetic determination) in common buckwheat. This phenomenon seems to be unbreakable by breeding methods. The authors aimed to determine whether stimulants commonly used in agriculture could increase the seed yield of this plant species. The aim of the work was to choose from eight different stimulants the most effective one that would improve the seed yield of two accessions of common buckwheat by increasing the efficiency of nectar production and reducing the number of empty seeds. The plants were sprayed at either the beginning of flowering or at full bloom. The content of sugars and amino acids was higher in the nectar produced at the beginning of flowering. The nectar of both lines included also polyamines. The level of sugars in the nectar increased mainly after spraying with the stimulants in the second phase of flowering. A positive correlation between the total amount of sugars and amino acids in the nectar and seed yield was found. All the stimulants used reduced the number of empty seeds in both accessions. Seed production in the PA15 line increased significantly under the influence of all stimulants used at the beginning of flowering, and the most effective were ASAHI SL and TYTANIT®.

Genetic Variation versus Morphological Variability in European Peatland Violets (Viola epipsila-V. palustris Group).

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.

Innate, High Tolerance to Zinc and Lead in Violets Confirmed at the Suspended Cell Level.

Many species of the Viola L. genus (violets) colonize areas with high concentrations of trace elements in the soil, e.g., nickel, cadmium, zinc, and lead. Although tolerance to heavy metals is a common phenomenon in violets, it is not clear whether this is the result of gradual microevolutionary processes as a part of the adaptation to the specific conditions, or whether the tolerance was inherited from the ancestor(s). We developed cell suspension cultures of five plant species: two non-metallophytes-Arabidopsis thaliana (Col-0) and Viola · wittrockiana, and three metallophytes-V. philippica, V. tricolor, and Silene vulgaris subsp. humilis for tolerance tests. The aim of the study was to measure the level of tolerance of violets in comparison with species from the other genera to verify the hypothesis of the high, innate tolerance of the former. We measured cell viability, non-enzymatic antioxidant content, and the accumulation of heavy metals after cell treatment with Zn or Pb. The results indicate they are innate and independent on the ecological status (metallophyte vs. non-metallophyte) and high in comparison with other species tolerance to Zn and Pb in violets. Viability of the cells after Zn and Pb (1000 µM) exposure for 72 h was the highest in violets. Antioxidant content, after heavy metal treatment, increased significantly, particularly in metallophyte violets, indicating their high responsivity to metals. In all species, lead was detected in the protoplasm of the cells, not in the vacuole or cell wall. All violets were characterized by the accumulation capacity of lead. Here, we clearly show that the physiological and biochemical studies conducted with the use of heavy metals on plant cells translate into the heavy metal tolerance of the species.

Re-introduction of an extinct population of Pulsatilla patens using different propagation techniques.

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.

Seasonal and Simultaneous Cleistogamy in Rostrate Violets (Viola, subsect. Rostratae, Violaceae).

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.

Insights into Plant Programmed Cell Death Induced by Heavy Metals-Discovering a Terra Incognita.

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.

Reducing Flower Competition for Assimilates by Half Results in Higher Yield of Fagopyrum esculentum.

Despite abundant flowering throughout the season, common buckwheat develops a very low number of kernels probably due to competition for assimilates. We hypothesized that plants with a shorter flowering period may give a higher seed yield. To verify the hypothesis, we studied nutrient stress in vitro and in planta and analyzed different embryological and yield parameters, including hormone profile in the flowers. In vitro cultivated flowers on media with strongly reduced nutrient content demonstrated a drastic increase in degenerated embryo sacs. In in planta experiments, where 50% or 75% of flowers or all lateral ramifications were removed, the reduction of the flower competition by half turned out to be the most promising treatment for improving yield. This treatment increased the frequency of properly developed embryo sacs, the average number of mature seeds per plant, and their mass. Strong seed compensation under 50% inflorescence removal could result from increased production of salicylic and jasmonic acid that both favor more effective pollinator attraction. Plants in single-shoot cultivation finished their vegetation earlier, and they demonstrated greater single seed mass per plant than in control. This result suggests that plants of common buckwheat with shorter blooming period could deliver higher seed yield.

Differences in the functioning of photosynthetic electron transport between metallicolous and non-metallicolous populations of the pseudometallophyte Viola tricolor.

The objective of this study was to assess the effect of metalliferous conditions on the functioning of photosynthetic electron transport in waste heap populations of a pseudometallophyte, Viola tricolor L. Measurements of chlorophyll a fluorescence and the absorbance changes at 830 nm enabled a non-invasive assessment of photosynthetic apparatus performance. This was complemented by the evaluation of the chlorophyll content. Low temperature chlorophyll fluorescence emission spectra were also recorded. Based on the OJIP test performed in situ, we demonstrated a disturbed condition of photosystem II (PSII) in three metalliferous populations in comparison with a non-metallicolous one. The combined effects of elevated concentrations of zinc, cadmium and lead in soil resulted in the decline of some parameters describing the efficiency and electron flow through PSII. The differences between waste heap populations seemed to be partly correlated with the concentration of heavy metals in the soil. The characteristic of electron transport at photosystem I (PSI) in the light-adapted state revealed increased values of PSI donor-side limitation (YND) and a declined PSI quantum efficiency (YI). It was also demonstrated that the waste heap conditions negatively affect the total chlorophyll content in leaves and led to an increased ratio of fluorescence emission at 77 K (F730/F685). The obtained data indicate that, regardless of the high adaptation of metallicolous populations, photosynthetic electron transport is hampered in V. tricolor plants at metal polluted sites.

From cells highly tolerant to Zn and Pb to fully fertile plants - Selection of tolerant lines with in vitro culture.

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.

Effects of High Temperature on Embryological Development and Hormone Profile in Flowers and Leaves of Common Buckwheat (Fagopyrum esculentum Moench).

Common buckwheat is a valuable crop, mainly due to the beneficial chemical composition of its seeds. However, buckwheat cultivation is limited because of unstable seed yield. The most important reasons for the low yield include embryo and flower abortion. The aim of this work is to verify whether high temperature affects embryological development in this plant species. The experiment was conducted on plants of a Polish cultivar 'Panda' and strain PA15, in which the percentage of degenerating embryo sacs was previously determined and amounted to 32% and 10%, respectively. The plants were cultivated in phytotronic conditions at 20 °C (control), and 30 °C (thermal stress). The embryological processes and hormonal profiles in flowers at various developmental stages (buds, open flowers, and wilted flowers) and in donor leaves were analyzed in two-month-old plants. Significant effects of thermal stress on the defective development of female gametophytes and hormone content in flowers and leaves were observed. Ovules were much more sensitive to high temperature than pollen grains in both genotypes. Pollen viability remained unaffected at 30 °C in both genotypes. The effect of temperature on female gametophyte development was visible in cv. Panda but not in PA15 buds. A drastic reduction in the number of properly developed embryo sacs was clear in open flowers at 30 °C in both genotypes. A considerable increase in abscisic acid in open flowers ready for fertilization may serve as a signal inducing flower senescence observed in the next few days. Based on embryological analyses and hormone profiles in flowers, we conclude that cv. 'Panda' is more sensitive to thermal stress than strain PA15, mainly due to a much earlier response to thermal stress involving impairment of embryological processes already in the flower buds.

Suspended cells of metallicolous and nonmetallicolous Viola species tolerate, accumulate and detoxify zinc and lead.

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.

How Does the Sweet Violet (Viola odorata L.) Fight Pathogens and Pests - Cyclotides as a Comprehensive Plant Host Defense System.

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.

Influence of salicylic acid pretreatment on seeds germination and some defence mechanisms of Zea mays plants under copper stress.

The study was focused on the influence of salicylic acid (SA) on maize seeds germination and on some physiological and biochemical processes in maize plants growing in the hydroponic culture under copper (Cu) stress. A significant influence of SA pretreatment on the advanced induction of the maize seeds metabolic activity and the level of the endogenous SA in germinated seeds and developing roots have been stated. Although, the ability of maize seeds to uptake SA and accumulate it in the germinated roots was confirmed, the growth inhibition of Cu-stressed maize seedlings was not ameliorated by SA seeds pretreatment. Cu-stressed plants exhibited a decrease in the photosynthetic pigment concentration and the increase in non-photochemical quenching (NPQ) - an indicator of an excess energy in PSII antenna assemblies lost as a heat. The amelioration effect of SA application was found only for carotenoids content which increased in stressed plants. It was also shown that maize roots growing in stress conditions significantly differed in the chemical composition in comparison to the roots of control plants, but the SA pretreatment did not affect these differences. On the other hand, it was found that SA seed pretreatment significantly influenced the ability of stressed plants to accumulate copper in the roots. It was stated that a higher level of exogenous SA application led to a lower accumulation of Cu ions in maize roots. Cu-stressed plants exhibited higher oxidative stress in roots than in leaves which was manifested as an increase in the concentration of hydrogen peroxide due to stress factor application. We observed an increase in catalase (CAT) activity in leaves of Cu-stressed plants which corresponded with a lower H2O2 content when compared with roots where the hydrogen peroxide level was higher, and the inhibition of the CAT activity was found. Furthermore, we found that the SA seed pretreatment led to a decrease in the H2O2 content in the roots of the Cu-stressed plants, but it did not influence the H2O2 level in leaves. The increase in hydrogen peroxide content in the roots of Cu-stressed plants correlated with a higher activity of the MnSODI and MnSODII isoforms. It was found that SA pretreatment caused a decrease in MnSODII activity accompanied by the decrease in H2O2 concentration. Achieved results indicated also that the changes in the chemical composition of the root tissue under copper stress constituted protection mechanisms of blocking copper flow into other plant organs. However, it might be assumed that the root tissue remodelling under Cu stress did not only prevent against the Cu ions uptake but also limited the absorption of minerals required for the normal growth leading to the inhibition of the plant development.

Divergent biology of facultative heavy metal plants.

Among heavy metal plants (the metallophytes), facultative species can live both in soils contaminated by an excess of heavy metals and in non-affected sites. In contrast, obligate metallophytes are restricted to polluted areas. Metallophytes offer a fascinating biology, due to the fact that species have developed different strategies to cope with the adverse conditions of heavy metal soils. The literature distinguishes between hyperaccumulating, accumulating, tolerant and excluding metallophytes, but the borderline between these categories is blurred. Due to the fact that heavy metal soils are dry, nutrient limited and are not uniform but have a patchy distribution in many instances, drought-tolerant or low nutrient demanding species are often regarded as metallophytes in the literature. In only a few cases, the concentrations of heavy metals in soils are so toxic that only a few specifically adapted plants, the genuine metallophytes, can cope with these adverse soil conditions. Current molecular biological studies focus on the genetically amenable and hyperaccumulating Arabidopsis halleri and Noccaea (Thlaspi) caerulescens of the Brassicaceae. Armeria maritima ssp. halleri utilizes glands for the excretion of heavy metals and is, therefore, a heavy metal excluder. The two endemic zinc violets of Western Europe, Viola lutea ssp. calaminaria of the Aachen-Liège area and Viola lutea ssp. westfalica of the Pb-Cu-ditch of Blankenrode, Eastern Westphalia, as well as Viola tricolor ecotypes of Eastern Europe, keep their cells free of excess heavy metals by arbuscular mycorrhizal fungi which bind heavy metals. The Caryophyllaceae, Silene vulgaris f. humilis and Minuartia verna, apparently discard leaves when overloaded with heavy metals. All Central European metallophytes have close relatives that grow in areas outside of heavy metal soils, mainly in the Alps, and have, therefore, been considered as relicts of the glacial epoch in the past. However, the current literature favours the idea that hyperaccumulation of heavy metals serves plants as deterrent against attack by feeding animals (termed elemental defense hypothesis). The capability to hyperaccumulate heavy metals in A. halleri and N. caerulescens is achieved by duplications and alterations of the cis-regulatory properties of genes coding for heavy metal transporting/excreting proteins. Several metallophytes have developed ecotypes with a varying content of such heavy metal transporters as an adaption to the specific toxicity of a heavy metal site.

Albanian violets of the section Melanium, their morphological variability, genetic similarity and their adaptations to serpentine or chalk soils.

Violets of the section Melanium from Albanian serpentine and chalk soils were examined for their taxonomic affiliations, their ability to accumulate heavy metals and their colonization by arbuscular mycorrhizal fungi (AMF). The sequence analysis of the ITS1-5.8S rDNA-ITS2 region showed that all the sampled six Albanian violets grouped between Viola lutea and Viola arvensis, but not with Viola tricolor. The fine resolution of the ITS sequences was not sufficient for a further delimitation of the Albanian violets within the V. lutea-V. arvensis clade. Therefore, the Albanian violets were classified by a set of morphological characters. Viola albanica, Viola dukadjinica and Viola raunsiensis from serpentine soils as well as Viola aetolica from a chalk meadow were unambiguously identified, whereas the samples of Viola macedonica showed high morphological variability. All the violets, in both roots and shoots contained less than or similar levels of heavy metals as their harboring soils, indicating that they were heavy metal excluders. All the violets were strongly colonized by AMF with the remarkable exception of V. albanica. This violet lived as a scree creeper in shallow serpentine soil where the concentration of heavy metals was high but those of P, K and N were scarce.

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

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

Metalliferous and non-metalliferous populations of Viola tricolor represent similar mode of antioxidative response.

Heavy metal-contaminated sites are excellent areas to examine the antioxidative machinery responsible for physiological adaptations of many plant species. Superoxide dismutase (SOD), guaiacol peroxide (GPX), ascorbate peroxide (APX), catalase (CAT) activity and hydrogen peroxide (H(2)O(2)) content were analyzed in leaves and roots of Viola tricolor (Viola) from contaminated soils ('Bukowno', 'Saturn', 'Warpie' heaps), and non-contaminated soil ('Zakopane meadow') to examine the level of oxidative stress and antioxidative response. In leaves, six isoforms of SOD were recognized. Roots possessed two additional bands, named manganese superoxide dismutase (MnSOD)-like form (MnSODI) and Cu/ZnSOD-like form (Cu/ZnSODIV). The H(2)O(2) content in leaves ranged from 554 to 5 098 micromol H(2)O(2)/gf.w. and was negatively correlated with CAT activity. The non-contaminated population was characterized by the lowest CAT activity combined with the highest H(2)O(2) concentration. Two isoforms of CAT, CAT-1 and CAT-2, were recognized in leaves of plants from non-contaminated and contaminated sites, respectively. In roots of individuals from two heaps ('Warpie' and 'Saturn'), two distinct bands for each CAT isoform were observed. A slower migrating band may be an aggregate, exhibiting CAT and MnSODs activities. Both peroxidases (APX and GPX) presented the same pattern of activity, depending on the organ, indicating that in leaves and roots APX and GPX were regulated in parallel. Differences in enzyme activities and H(2)O(2) content between plants from different contaminated sites were statistically significant, but were tightly maintained at a very similar level. Prolonged and permanent heavy metal stress evoked a very similar mode of antioxidative response in specimens of analyzed metalliferous populations not causing measurable oxidative stress. Thus, our results clearly indicate that V. tricolor is a taxon well adapted to heavy metal-contaminated soils, and that differences in enzyme activities and H(2)O(2) content result from adjustment of plants to a variety of conditions.