Ewa Muszyńska

Phytostabilization of Zn-Pb ore flotation tailings with Dianthus carthusianorum and Biscutella laevigata after amending with mineral fertilizers or sewage sludge

Zinc-lead mining wastes remain largely unvegetated and prone to erosion for many years because of phytotoxic levels of residual heavy metals, low nutrient status and poor physical structure. The optimal solution for these areas is to restore plant cover using species which spontaneously appear on the spoils. These species are adapted to the conditions of tailings, and their establishment will promote further vegetation by increasing soil organic matter and development of a soil system capable of supporting the nutrient and water requirements of plants and microoorganisms. The potential of Dianthus carthusianorum and Biscutella laevigata to stabilize mine spoils was analysed in a three-year pot experiment. Post-flotation wastes accumulated after Zn and Pb recovery from ores, were collected from tailings and used as a substrate for plant growth. Seeds for seedling production were collected from plants growing spontaneously on mine tailings. Prior to the establishment of the three-year pot experiment, the substrate was amended with fertilizer NPK or municipal sewage sludge, supplemented with K2O (SS). Substrate samples were collected for chemical analyses, dehydrogenase and urease activities measurements each year at the end of the growing season. The plants were harvested three years after the amendments. Both tested plant species were equally suitable for revegetation of the tailings. The amendment including both SS and NPK resulted in an increase of Corg, Nt, available P, K, Mg contents, an increase of dehydrogenase (DHA) and urease activities and a decrease in the concentrations of the soluble forms of Zn, Pb and Cd. However, nutrient content, DHA activity and plant biomass were higher with SS than NPK addition. NPK application enhanced the substrate properties after the first growing season, while positive effects of SS use were still observed after three years. A longer-lasting positive effect of SS than NPK application was probably due to the high organic matter content in SS, which was gradually decomposing and releasing nutrients.

Comparative Assessment of Response to Cadmium in Heavy Metal-Tolerant Shrubs Cultured In Vitro

Two species of Pb-adapted shrubs, Alyssum montanum and Daphne jasminea, were evaluated in vitro for their tolerance to elevated concentrations of cadmium. Shoot cultures were treated with 0.5, 2.5, and 5.0xa0μM CdCl2 for 16xa0weeks and analyzed for their organogenic response, biomass accretion, pigment content, and macronutrient status. Cadmium accumulation and its root-to-shoot translocation were also determined. In both species, rooted microplantlets, suitable for acclimatization, were obtained in the presence of Cd applied as selection agent. In A. montanum, low and moderate dose of Cd stimulated multiplication, rooting, and biomass production. Growth tolerance index (GTI) in Cd-treated shoots ranged from 120 to 215%, while in the roots 51–202%. In turn, in Cd-treated D. jasminea proliferation and rooting were inhibited, and GTI for shoots decreased with increasing doses of Cd. However, roots exposed to Cd had higher biomass accretion. Both species accumulated Cd in developed organs, and its content increased with increasing CdCl2 dose. Interestingly, D. jasminea accumulated higher amounts of Cd in the roots than A. montanum and immobilized this metal in the root system. On the contrary, A. montanum translocated some part of accumulated Cd to the shoots, but with low efficiency. In the presence of Cd, A. montanum maintained macronutrient homeostasis and synthesized higher amounts of phytosynthetic pigments in the shoots. D. jasminea accumulated root biomass, immobilized Cd, and restricted its translocation at the expense of nutrient balance. Considering remediation potential, A. montanum could be exploited in phytoextraction, while D. jasminea in phytostabilization of polluted substrate.

From laboratory to field studies – The assessment of Biscutella laevigata suitability to biological reclamation of areas contaminated with lead and cadmium

The aim of the work was to evaluate the usefulness of the in vitro multiplication of Biscutella laevigata calamine ecotype for in situ reclamation of post-flotation wastes polluted with Pb and Cd. The experiment was conducted on three steps: (i) plant shoots production under in vitro condition, (ii) establishment of the material in greenhouse experiment, and finally (iii) field cultivation directly on the mining-waste heap of Olkusz Ore-Bearing Region, Poland. This region is known to be one of the most chemically-degraded area in central Europe. The laboratory-set in vitro analysis enabled to obtain the high-quality plant shoots, which multiply the most effectively (with growth tolerance index 130-150%) on medium containing 5.0μM CdCl2 and 0.5mM Pb(NO3)2. These plants were used for the next two ex vitro experiments. Several biometric and physiological analysis (i.e. of photosystem II activity Fv/Fm and PI, photosynthetic pigment contents) were done to indicate plant physiological status during these experiments. The main novelty of the work was to prove that in vitro-multiplied shoots of B. laevigata - the representative of native flora from Olkusz Ore-Bearing Region - can be successfully implemented in situ for the restoration of these degraded area. Moreover, the addition of sewage sludge as a source of organic compounds significantly improved plants growth and development what is especially important due to the lack of other legal solutions for the management of the sewage sludge in some countries.

Recent strategies of increasing metal tolerance and phytoremediation potential using genetic transformation of plants

Avoidance and reduction of soil contamination with heavy metals is one of the most serious global challenges. Nowadays, science offers us new opportunities of utilizing plants to extract toxic elements from the soil by means of phytoremediation. Plant abilities to uptake, translocate, and transform heavy metals, as well as to limit their toxicity, may be significantly enhanced via genetic engineering. This paper provides a comprehensive review of recent strategies aimed at the improvement of plant phytoremediation potential using plant transformation and employing current achievements in nuclear and cytoplasmic genome transformation. Strategies for obtaining plants suitable for effective soil clean-up and tolerant to excessive concentrations of heavy metals are critically assessed. Promising directions in genetic manipulations, such as gene silencing and cis- and intragenesis, are also discussed. Moreover, the ways of overcoming disadvantages of phytoremediation using genetic transformation approachare proposed. The knowledge gathered here could be useful for designing new research aimed at biotechnological improvement of phytoremediation efficiency.

In vitro multiplication of Dianthus carthusianorum calamine ecotype with the aim to revegetate and stabilize polluted wastes

Abandoned metalliferous wastes can be spontaneously colonized by specialized species or ecotypes, and therefore representatives from such populations might be exploited in phytoremediation. Thus, this study was focused on determining the conditions for culture initiation and elaborating the propagation protocol of wild calamine ecotype of Dianthus carthusianorum. The proper propagation medium proved modified MS enriched with 1.0xa0mg/L 2iP and 0.2xa0mg/L IAA. The massive majority (93%) of microplantlets were successfully transferred to ex vitro conditions. Micropropagated calamine ecotype of D. carthusianorum has proved to be useful for phytoremediation application. The obtained plants experimentally cultivated on post-flotation wastes generated during the process of zinc-lead ore enrichment were monitored, and compared with specimens of the population obtained as a result of seed sowing. Plants propagated through tissue culture grew better, developed faster and more abundantly bloomed in comparison with the generatively propagated control material. This is one of the few reports concerning the possibility of using in vitro technique for effective production of plant material ready to be used in chemically degraded area.

Organic amendments enhance Pb tolerance and accumulation during micropropagation of Daphne jasminea

The study investigated the effects of organic amendments: pineapple pulp (PP) and agar hydrolyzate (AH), on micropropagation and Pb bioaccumulation and tolerance in a woody shrub Daphne jasminea cultured in vitro. The amendments were analyzed for their content of carbohydrates, phenolic acids, and phytohormones and added at a dose of 10xa0mLxa0L−1 to the medium containing 1.0xa0mM lead nitrate. Micropropagation coefficient increased by 10.2–16.6xa0% in PP and AH variants, respectively. Growth tolerance index increased by 22.9–31.8xa0% for the shoots and by 60.1–82.4xa0% for the roots. In the absence of Pb, the additives inhibited multiplication and growth of microplantlets. PP and AH facilitated Pb accumulation in plant organs, especially in the roots. PP enhanced bioconcentration factor and AH improved Pb translocation to the shoots. Adaptation to Pb was associated with increased accumulation of phenolics and higher radical scavenging activity. Medium supplementation, particularly with AH, enhanced antiradical activity of Pb-adapted lines but reduced the content of phenolic compounds. The study results indicated that supplementation with organic amendments may be beneficial in in vitro selection against lead toxicity.

Differential Tolerance to Lead and Cadmium of Micropropagated Gypsophila fastigiata Ecotype

In vitro techniques may provide a suitable tool for effective propagation and conservation of plant species representing various ecological niches. The elaboration of such protocols is also prerequisite for selection of heavy-metal-tolerant plant material that could be afterwards used for restoration or remediation of polluted sites. In this study, culture protocol for Gypsophila fastigiata propagation was developed. The highest multiplication coefficient, which reached 6.5, and the best growth parameters were obtained on modified MS medium supplemented with 1.0xa0mgxa0L−1 2iP and 0.2xa0mgxa0L−1 IAA. The obtained cultures were treated with different concentrations of lead nitrate (0.1, 0.5, and 1.0xa0mM Pb(NO3)2) or cadmium chloride (0.5, 2.5, and 5.0xa0μM CdCl2). The growth parameters, photosynthetic pigments, and phenolic compound content were examined in order to evaluate whether tested metal salts can have an adverse impact on studied culture. It was ascertained that Pb ions induced growth disturbances and contributed to shoot wither. On the contrary, the proliferative shoot cultures were established on media containing Cd ions and the multiplication coefficients and shoot length increased on all media enriched with CdCl2. Chlorophylls and carotenoid contents were negatively affected by application of 5.0xa0μM of cadmium; nevertheless, in shoots treated with 2.5xa0μM CdCl2, increased accumulation of photosynthetic pigments occurred and their amount was similar to untreated culture. Adaptation to Cd was associated with stimulation of phenolic compound synthesis. Hence, we have reported on unambiguous positive result of in vitro selection procedure to obtain vigorous shoot culture tolerant to cadmium.

The acclimatization strategies of kidney vetch (Anthyllis vulneraria L.) to Pb toxicity

Kidney vetch (Anthyllis vulneraria L.) is a well-known Zn hyperaccumulator. Zn often occurs with Pb in one ore; thus, plants inhabiting waste dumps are exposed not only to Zn but also to Pb toxicity. While the response of kidney vetch to Zn toxicity is relatively well known, the Pb survival strategy of Anthyllis vulneraria has not been the subject of investigations. The aim of presented research was to determine the survival strategy of kidney vetch exposed to high lead concentrations. Shoot explants of a calamine kidney vetch ecotype were placed on agar media containing 0.0, 0.5, 1.0, and 1.5xa0mM Pb. Morphological, physiological, and biochemical responses, in particular photosynthetic apparatus of plantlets, were examined. The most pronounced changes were observed in plants grown on media supplemented with 1.5xa0mM Pb after 8xa0weeks of culture. Increased dry weight and high lead accumulation were observed in roots. Similarly, in shoots, increased dry weight and a decreased number of newly formed shoots were recorded. The accumulation of lead was many times lower in shoots than in roots. In leaf cells’ ultra-structure, looser arrangement of chloroplast thylakoid grana was observed. Despite the decrease in chlorophyll a and carotenoid content, the photosynthetic apparatus remained efficient due to the lack of photoinhibition and increased electron transport rate beyond photosystem II (PSII). For the first time, an acclimatization mechanism based on maintaining the high efficiency of photosynthetic apparatus resulting from increasing of electron transport rate was described.

Evaluation of Scabiosa ochroleuca L. vitality after introduction on post-flotation wastes

Abstract The aim of this study was to evaluate the Scabiosa ochroleuca suitability for growth on heavy metals contaminated substrate obtained as a result of Zn–Pb ores exploitation and their processing. Three variants of cultivation were tested in vegetative hall conditions: post-flotation wastes (control), wastes enriched with either mineral fertilisation or with sewage sludge. The possibility of future direct use of the studied species on post-flotation settling pond was determined on the basis of biometric measurements and analysis efficiency of photosynthetic apparatus, that is, pigments content and chlorophyll a fluorescence. The results showed that untreated wastes negatively influenced on plants morphological and physiological status. On the contrary, the application of sewage sludge improved growth and development of cream scabious. Thus, not only the selection of proper plant material, tolerant to toxic levels of heavy metals and other hostile conditions, but also the waste treatment are crucial when metalliferous areas are to be reclaimed.

Studies on lead and cadmium toxicity in Dianthus carthusianorum calamine ecotype cultivated in vitro

Information on metallophytes during reclamation of land contaminated with heavy metals is sparse. We investigated the response of D.xa0carthusianorum calamine ecotype to Pb and Cd stress. We focused on inxa0vitro selection of tolerant plant material for direct use in chemically degraded areas. Shoot cultures were treated with various concentrations of Pb or Cd ions. Plantlet status was estimated as micropropagation efficiency, growth tolerance index (GTI) and through physiological analysis. Moreover, determination of plant Pb, Cd and other elements was performed. The application of Pb(NO3 )2 resulted in stronger growth inhibition than application of CdCl2 . In the presence of Pb ions, a reduction was observed of both, the micropropagation coefficient to 1.1-1.8 and the GTI to 48%. In contrast, Cd ions had a positive influence on tested cultures, expressed as an increase of GTI up to 243% on medium enriched with 1.0xa0μm CdCl2 . Moreover, photosynthetic pigment content in shoots cultivated on media with CdCl2 was higher than in control treatment. The adaptation to Cd was associated with decreased accumulation of phenols in the order: 0.0xa0μmxa0>xa01.0xa0μm > 3.0xa0μmxa0>xa05.5xa0μm CdCl2 . It seems that high tolerance to Cd is related to K uptake, which is involved in antioxidant defence. This work presents an innovative approach to the impact of Cd ions on plant growth and suggests a potential biological role of this metal in species from metalliferous areas.