Long-term organogenic callus cultivation of Ranunculus illyricus L.: a blueprint for sustainable ex situ conservation of the species in urban greenery.

The growing trend of introducing wild plant species into urban environments necessitates the identification of novel species adapted to prevailing conditions. A promising reservoir of such species may be xerothermic communities where Ranunculus illyricus occurs. This study aimed to establish a micropropagation protocol for R. illyricus using indirect organogenesis. The protocol includes initiation of culture from various explants, callus proliferation, shoot regeneration, multiplication, and concurrent rooting. Callus appeared on most types of vegetative explants tested, but stolons were considered the best due to their good availability, high disinfection (85%), and robust callus production (maximum increase - 363.1%). The growth rate of the callus fresh matter (CFM) obtained from stolons was calculated. Greater CFM was obtained on the medium with the supplemented picloram 8.0 mg L- 1 with kinetin 5.0 mg L- 1 and in second part of experiment on medium with the addition of 2,4-D (2,4-dichlorophenoxyacetic acid) 2.0 mg L- 1 alone or picloram 6.0 mg L- 1 with kinetin 8.0 mg L- 1. Shoot organogenesis was observed on macronutrients B5 (Gamborg medium), micronutrients MS (Murashige and Skoog) medium with the addition of 2.0 mg L- 1 IBA (indole-3-butyric acid) and 4.0 mg L- 1 BAP (6-benzylaminopurine). To document the process of callus differentiation, microscopic preparations were prepared. Subsequently, the regenerated plants underwent acclimatisation and their growth in an ex situ collection was monitored over three growing seasons. In particular, in vitro-origin plants exhibited developmental patterns similar to those of their seed-origin counterparts. The incorporation of R. illyricus into urban landscapes not only increases aesthetic appeal, but also ensures the preservation of valuable genetic resources for this rare species, potentially contributing to effective ex situ conservation in the future. This marks the first scientific report on in vitro cultures of R. illyricus.

Do chromium-resistant bacterial symbionts of hyperaccumulator Callitriche cophocarpa support their host in phytobial remediation of water?

Callitriche cophocarpa Sendtn. is a macrophyte widely distributed in aquatic systems of the temperate climate zone and a known hyperaccumulator of chromium. Ten pure symbiotic bacterial isolates of C. cophocarpa were obtained and identified. Three of the isolates showed the highest resistance to Cr(VI): Microbacterium sp. (Ct1), Aeromonas sp. (Ct3) and Acinetobacter sp. (Ct6). Acinetobacter sp. (Ct6) was able to survive up to a concentration of 104 mg/L (2 mM). The isolates were also able to effectively detoxify Cr(VI) by reducing it to Cr(III). We tested whether inoculation of plants with a consortium consisting of Ct1, Ct3 and Ct6 affects: (1) the phytoextraction of chromium from leachates, (2) the physiological state of plants after Cr(VI) treatment. The solutions were landfill leachates and contained 10.7 mg/L of Cr(VI) - an amount 530 times exceeding the legal limits. We influenced the plants with Cr in two steps, each lasting for 10 days, first using mature shoots and then apical ones. The highest Cr content concomitant with the highest bioconcentration factor (BCF) were found in the inoculated plants: 1274 and 119 mg/kg dry mass (d.m.), respectively. The physiological status of the plants was assessed by biometric tests and advanced chlorophyll fluorescence analyses. The photosynthetic activity of mature shoots was influenced by Cr(VI) more negatively than that of young apical shoots. The inoculation with the bacterial consortium significantly reduced the negative effect of Cr(VI) on mature organs. In some cases the inoculated mature plants exhibited photosynthetic activity that was even higher than in the control plants. The results unequivocally show a beneficial effect of C. cophocarpa inoculation with the tested isolates resulting in a significant improvement of the phytoremediation properties of this aquatic chromium hyperaccumulator.

Exploring a unique water ecosystem under long-term exposure to hexavalent chromium - An in situ study of natural diatom (Bacillariophyceae) communities.

This paper reports the third in a series of three studies of a unique aquatic phytobial consortium that developed in a polluted ditch situated near an old chromium waste landfill. The ditch is a specific ecological niche having increased loads of several chemical compounds, including extreme hexavalent chromium concentrations up to two thousand times the allowed limit (0.02 mg dm-3 in Poland) in the more polluted section B; the moderate concentrations in section A are twice the limit. We focus on the microscopic algae group of diatoms, one of the most important components of the phytobial consortium, and continue our novel attempt to analyze the bioremediation potential of the entire consortium under those environmental conditions. We used numerical methods to analyze differences in diatom biodiversity between sections A and B, and assessed the relations between diatoms and selected water chemistry properties, including hexavalent chromium, chlorides and sulphates, the latter two known to positively influence the resistance of algae to the impact of hexavalent chromium. We noted 37 diatom taxa in section A of the ditch and 30 in section B. The most frequently observed diatoms were cosmopolitan taxa and/or characteristic taxa of saline waters. Sulphates were the most important factor influencing the diatom composition in the ditch, explaining 52% of the total variability, followed by chlorides (30%) and hexavalent chromium (9%). Gomphonema acuminatum, Melosira varians and Nitzschia frustulum var. frustulum were found to be most resistant to hexavalent chromium and were selected for further experimental studies on their biotechnological usefulness.

Reproductive Biology of Dry Grassland Specialist Ranunculus illyricus L. and Its Implications for Conservation.

Ranunculus illyricus, a component of xerothermic grasslands, is a declining species and deserves active conservation treatments in many countries preceded by studies on the biology of its reproduction. So far, our knowledge of R. illyricus, a species with two modes of reproduction, has been fragmentary. The purpose of the studies presented here was to describe the annual development cycle of R. illyricus with particular emphasis on the production of underground tuber clusters that serve as vegetative propagation. Based on three-year-long observations in an ex situ collection, the efficiency of vegetative propagation was estimated and compared with the efficiency of generative propagation. It was found that in 3 years the best clones could produce up to 57 progeny clusters followed by flowering specimens in the first season. Meanwhile, the high potential for generative reproduction was suppressed by many limitations including fruit setting, the germination capacity of seeds, seedling survival rate, and additionally, the first flowering plant was observed only in the third year. It seems that the efficiency of vegetative propagation of this species can be higher than the efficiency of generative propagation. Moreover, vegets bloomed in the first year after emergence, whereas the first plant of generative origin was observed to bloom only after 3 years. A large proportion of individuals of vegetative origin can negatively affect the genetic diversity of the population but their survival rate against competing plants is higher. To enhance the existing populations or to create new ones, it would be best to use plants derived from clonal propagation of genets carried out in ex situ conditions.

The Effectiveness of the Sexual Reproduction in Selected Clonal and Nonclonal Species of the Genus Ranunculus.

Generative processes have been evaluated in six European buttercup species in order to verify the hypothesis that the reproduction efficiency of clonal species is lower than that of nonclonal ones. The study covered common species (Ficaria verna, Ranunculus auricomus, R. bulbosus, R. cassubicus, R. lanuginosus) and the endangered R. illyricus. The following properties have been assessed: pollen viability (staining method), pollen grain germination and the pollen-tube elongation in pistil tissues (fluorescence microscopy), seed formation efficiency, seed viability (tetrazolium test) and germination ability by introducing factors interrupting dormancy (low temperature and gibberellin application). Additionally, the pistil morphology was documented for R. bulbosus, R. illyricus and R. cassubicus using SEM techniques. It was demonstrated that the reproductive efficiency, expressed as the production of viable seeds able to germinate, was significantly higher in the species reproducing sexually (especially in R. lanuginosus) compared to the clonal ones. However, the complexity observed leads to separation of an additional group (cluster) of apomictic species: R. auricomus and R. cassubicus, distinguished by the lowest pollen viability and a low ability of the seeds to germinate. In the vegetatively reproducing R. illyricus, the seed formation efficiency was just 13.2% despite the having highest number of pistils in its flowers. The developed seeds of this species observed in our experiment were viable, but in general effective methods to stimulate their germination have not been proposed yet. Here, the first comparative study concerning the biology of sexual reproduction of R. illyricus is presented in the context of its decreasing distribution in natural habitats.

Discovery of 5-{2-[5-Chloro-2-(5-ethoxyquinoline-8-sulfonamido)phenyl]ethynyl}-4-methoxypyridine-2-carboxylic Acid, a Highly Selective in Vivo Useable Chemical Probe to Dissect MCT4 Biology.

Due to increased lactate production during glucose metabolism, tumor cells heavily rely on efficient lactate transport to avoid intracellular lactate accumulation and acidification. Monocarboxylate transporter 4 (MCT4/SLC16A3) is a lactate transporter that plays a central role in tumor pH modulation. The discovery and optimization of a novel class of MCT4 inhibitors (hit 9a), identified by a cellular screening in MDA-MB-231, is described. Direct target interaction of the optimized compound 18n with the cytosolic domain of MCT4 was shown after solubilization of the GFP-tagged transporter by fluorescence cross-correlation spectroscopy and microscopic studies. In vitro treatment with 18n resulted in lactate efflux inhibition and reduction of cellular viability in MCT4 high expressing cells. Moreover, pharmacokinetic properties of 18n allowed assessment of lactate modulation and antitumor activity in a mouse tumor model. Thus, 18n represents a valuable tool for investigating selective MCT4 inhibition and its effect on tumor biology.

Natural community of macroalgae from chromium-contaminated site for effective remediation of Cr(VI)-containing leachates.

The natural macroalgal community, which developed in the unique and extremely Cr(VI)-polluted aquatic reservoir situated near a historical chromium-waste landfill, was studied in order to recognize the main mechanisms of Cr(VI) detoxification by the algal species. The conducted taxonomic analysis revealed mixed composition of the filamentous forms of algae and showed that three species of Tribonema, namely T. vulgare, T. microchloron and T. viride, which have not been studied before with regard to the mechanisms of Cr(VI) removal, are likely responsible for the effective bioremediation of this highly Cr(VI)-polluted habitat. The studied algal community, with the ability to grow in extremely high concentrations of Cr(VI), i.e. up to ca. 6150 times the upper limit for surface water, exhibited hyperaccumulative properties for chromium (max 16230 mg/kg dry weight) under the given environmental conditions. We found that the main mechanism of Cr(VI) detoxification was reduction followed by Cr(III) biosorption - feasibly by ion exchange and complexation mechanisms - and that the excellent efficiency of chromium reduction under the given, unfavorable weakly alkaline conditions indicates the biological origin of this process. It was concluded that the examined reservoir inhabited by the algal community can be used, after some modifications, as a simple cost-effective "bioreactor" allowing the reduction of chromium concentration to the desired level. Moreover, the conducted studies are also essential to obtain in-depth knowledge and should also be helpful in the relevance of the community for its further application as a potential biosorbent of Cr(VI) on a global scale.

Unique biocenosis as a foundation to develop a phytobial consortium for effective bioremediation of Cr(VI)-polluted waters and sediments.

This paper analyzes a unique, aquatic phytobial biocenosis that has been forming naturally for over 20 years and operating as a filter for Cr(VI)-polluted groundwater. Our study presents a thorough taxonomic analysis of the biocenosis, including filamentous algae, vascular plants, and microbiome, together with the analysis of Cr accumulation levels, bioconcentration factors and other environmentally-significant parameters: siderophore production by bacteria, biomass growth of the plants or winter hardiness. Among 67 species identified in the investigated reservoir, 13 species were indicated as particularly useful in the bioremediation of Cr(VI)-polluted water and sediment. Moreover, three species of filamentous algae, Tribonema sp., and three easily culturable bacterial species were for the first time shown as resistant to Cr concentration up to 123 mg/dm3, i.e. 6150 times over the permissible level. The work presents a modern holistic phytobial consortium indispensable for the remediation of Cr(VI)-contaminated aquatic environment in temperate zones worldwide.

Sourcing and Propagation of Pontechium maculatum for Horticulture and Species Restoration.

Pontechium maculatum, a species of ornamental, apicultural, health and medicinal value, is threatened in some Central European countries including Poland. Its propagation using seeds or in vitro techniques is needed for multiple applications including conservation. Generative propagation efficacy of P. maculatum plants representing different genetic resources (received from botanical gardens in Germany and in Poland) propagated from seeds or in tissue culture was assessed. Moreover, an efficient technique of propagation of P. maculatum using in vitro shoot culture from seedlings was elaborated for the first time. The highest propagation efficacy was noted for German plants of seed origin. The ability of seeds to germinate was similar for all plants; however, seeds were in a state of dormancy, which was broken by GA3. After two years of storage, the seeds still retained the ability to germinate though seeds from propagated in vitro plants germinated more poorly than those from seed-originated plants. The ploidy assessment showed that some in vitro-origin plants had altered DNA content. The results indicate that efficacy of generative propagation of P. maculatum is resource dependent. Furthermore, results suggest that cultivation in vitro influenced some generative features of examined species, which makes this way of P. maculatum propagation a valuable source of genetic variation and a potential breeding tool.

The use of Callitriche cophocarpa Sendtn. for the reclamation of Cr-contaminated freshwater habitat: benefits and limitations.

This work is the first attempt to evaluate suitability of Callitriche cophocarpa Sendtn. (water-starwort) to remove Cr under real-world conditions. Our earlier laboratory-scale studies demonstrated outstanding hyperaccumulation properties of this aquatic higher plant (macrophyte) toward chromium in solution. We introduced C. cophocarpa plants into the watershed with sediments heavily polluted (on average 1400 mg/kg d.w. of Cr) by a tannery. The plants grew vigorously and exhibited no physiological or anatomical disorders. Based on chemical fractionations of bottom sediments, we found low Cr bioavailability. The element was strongly associated with the sediments and could be classified into the following fractions (%): oxidizable III (68.2) > residual IV (28.8) > reducible II (1.6) > exchangeable I (1.4). Despite this, Cr content in plant organs at the contaminated sites was 33 up to 83 times greater than in the control leaf/stem and roots, respectively. Altering redox potential during, i.e., sediment deposition on land may change chemical forms of bound metals in a solid phase, and thus further increase Cr phytoextraction by plants. With this in mind, we concluded that the species, being an outstanding Cr accumulator under laboratory conditions, can be useful in the reclamation of Cr-polluted sediments under controlled, oxidizing conditions.

Can Ceylon Leadwort (Plumbago zeylanica L.) Acclimate to Lead Toxicity?-Studies of Photosynthetic Apparatus Efficiency.

Ceylon leadwort (Plumbago zeylanica) is ornamental plant known for its pharmacological properties arising from the abundant production of various secondary metabolites. It often grows in lead polluted areas. The aim of presented study was to evaluate the survival strategy of P. zeylanica to lead toxicity via photosynthetic apparatus acclimatization. Shoots of P. zeylanica were cultivated on media with different Pb concentrations (0.0, 0.05, and 0.1 g Pb∙l-1). After a four-week culture, the efficiency of the photosynthetic apparatus of plants was evaluated by Chl a fluorescence measurement, photosynthetic pigment, and Lhcb1, PsbA, PsbO, and RuBisCo protein accumulation, antioxidant enzymes activity, and chloroplast ultrastructure observation. Plants from lower Pb concentration revealed no changes in photosynthetic pigments content and light-harvesting complex (LHCII) size, as well as no limitation on the donor side of Photosystem II Reaction Centre (PSII RC). However, the activity and content of antioxidant enzymes indicated a high risk of limitation on the acceptor side of Photosystem I. In turn, plants from 0.1 g Pb∙l-1 showed a significant decrease in pigments content, LHCII size, the amount of active PSII RC, oxygen-evolving complex activity, and significant remodeling of chloroplast ultrastructure indicated limitation of PSII RC donor side. Obtained results indicate that P. zeylanica plants acclimate to lead toxicity by Pb accumulation in roots and, depending on Pb concentration, by adjusting their photosynthetic apparatus via the activation of alternative (cyclic and pseudocyclic) electron transport pathways.