The improving influence of laser stimulation on phytoremediation capabilities of selected Silene vulgaris ecotypes

The aim of the paper is to improve the phytoremediation features of the metallophyte Silene vulgaris through photo-stimulation of seeds using a semi-conductive laser. Seeds of two Silene vulgaris ecotypes were used in the experiment. One type of seeds – “Wiry” ecotype – originated from a site contaminated with heavy metals (a serpentinite waste heap), and the other ecotype – “Gajków” – was collected on a site with naturally low heavy metal content. The seeds of both types were preconditioned with laser light with previously fi xed doses: C(D0), D1, D3, D5, D7, D9. The basic radiation dose was 2.5·10-1 J∙cm-2. The soil for the experiment was serpentinite weathering waste. The seeds and plants were cultivated in the controlled conditions of a climatic chamber. Laser light indeed stimulated seed germinative capacity but better effects were obtained in “Wiry” ecotype, originating from a location contaminated with heavy metals. In the case of morphological features, a signifi cant differentiation of stem length was found for different ecotypes, dosages and the interactions of these factors. The study showed a strong infl uence of laser radiation on selected element concentrations in above-ground parts of Silene vulgaris, though “Wiry” ecotype clearly accumulated more heavy metals and magnesium than the “Gajków” ecotype. 80 A. Koszelnik-Leszek, H. Szajsner, M. Podlaska General characteristics of selected Silene vulgaris ecotypes and their habitats The “Gajków” (non metallicolous) ecotype originates from a natural habitat in the village Gajków located to the south-east of Wrocław (51°3′25.021 ̋N, 17°11′9.415 ̋E). The “Wiry” (serpentine) ecotype grows on a small post-mining serpentinite heap near a village called Wiry located close to the western slopes of Mount Ślęża (50°50′12.773 ̋N, 16°37′3.724 ̋E). Serpentinites are peculiar rocks whose occurrence in Poland is limited to Lower Silesia (Żołnierz 2007). The particular chemistry of serpentinites, formed through transformed ultra-alkali intrusive rocks, results in unique soil rich in magnesium and low in calcium (low Ca:Mg ratio), and additionally rich in Ni, Cr and Co in values exceeding noted values from other Lower Silesian soils (Żołnierz 2007). Silene vulgaris (Moench) Garcke is a perennial plant from the Caryophyllaceae family. The species is commonly found in Europe, northern Africa, Asia and both Americas (Koszelnik-Leszek and Bielecki 2013). In Poland the species occurs on grasslands, fi elds and in forests, but it can also be found in synanthropic plant communities as an element of ruderal habitats such as serpentinite or calamine heaps or other post-mining areas (Koszelnik-Leszek and Bielecki 2013). Silene vulgaris is a bioindicator of heavy metal contamination and its presence has been recorded in areas both naturally rich in heavy metals and contaminated by human activity (Bratteler et al. 2002, Kazakou et al. 2010, Koszelnik-Leszek and Bielecki 2013, Nadgórska-Socha et al. 2011, Rostański et al. 2015, Wierzbicka and Panufnik 1998). The literature provides examples of the species’ unique adaptation capabilities leading to the formation of separate ecotypes adapted to extreme habitat conditions. Apart from Silene vulgaris ecotypes immune to lead and zinc, there are also ones able to tolerate an abundance of copper and nickel (Koszelnik-Leszek and Bielecki 2013). Pot growth test The laboratory experiment using a semi-conductive laser (model CTL-1106MX with a power of 200 mW and a wavelength of 670 nm) consisted in pre-sowing irradiation of seed conditioning of selected Silene vulgaris ecotype seeds with previously determined radiation dosages. The dosages were as follows: single – (D1), three – (D3), fi ve – (D5), seven – (D7) and nine-fold (D9) fold basic dosage of 2.5·10-1 J∙cm-2. The duration of individual exposure was 4.1 min. The control group C (D0) consisted of seeds without any dosages. The irradiated seeds of both Silene vulgaris ecotypes were then planted in pots with 1 kg of weathering waste originating from the serpentinite heap in Wiry (20 pieces per pot). Each combination of ecotype and dosage was performed in three repetitions. Plants were cultivated in the control conditions of a climatic chamber (Sanyo model MLR-351). Germination capacity and rate (ISTA 2008) were determined for every dosage and ecotype. After 6 weeks of the experiment the height and width of leaf blades were calculated, and after 8 weeks leaf blades were collected and secured for further analyses, which determined the infl uence of radiation dosages on metal accumulation and growth stimulation. The collected samples (above-ground plant parts) were subjected to dry mineralization. The contents of Ni, Cr, Co, Zn and Mg were determined using the AAS method on SpectrAA 220 Fast Sequential equipment. The soil used in the experiment was weathering waste from a serpentinite heap. Before the experiment the material was analyzed for selected parameters. Soil pH was noted potentiometrically at 1 mol KCl·dm-3. Phosphorus and potassium were recorded using Egner-Riehm method, magnesium by Schachtschabel method. Total and available heavy metal content was determined using the method of Atomic Absorption Spectrophotometry with the use of SpectrAA 220 Fast Sequential equipment. Statistical analysis. Seed quality and morphological features The obtained results were analyzed statistically (Statistical Tool for Agricultural Research 2012) using two way analysis of variance with factor I as the two selected ecotypes of Silene vulgaris and factor II as the radiation dosages. Fisher-Snedecor distribution was used to determine the relevance of the studied variance sources. Duncan’s new multiple test range was used to determine homogeneous groups. The chemical composition of plant material Statistical analyses were conducted using Statistica ver. 12 software (StatSoft Inc. 2014). Normal distribution was checked using Kruskal-Wallis test. Variance analysis and Fisher’s exact test were used to determine the differences in element concentrations in the selected Silene vulgaris ecotypes. Variance homogeneity was determined using Levene’s test. The data for which normal distribution or variance homogeneity were not obtained were analyzed using Kruskal-Wallis test. Results and discussion Serpentinite soil used in the pot growth experiment was characterized by high heavy metal content (Tab. 1). The concentrations of Ni, Cr, Co and Zn (both total and available) exceeded mean values of these elements for Poland and Central Europe (Kabata-Pendias 2011). The values were, however, typical of Polish serpentinite occurrences (Żołnierz 2007). The analysis showed that material from the excavation heap was characterized by a particularly low concentrations of phosphorus and potassium (for Poland) and, at the same time, particularly high or even toxic concentration of magnesium (again, for Poland). The recorded pH (KCl) of 7.8 is typical of serpentinite habitats in Lower Silesia (Żołnierz 2007). Laser radiation as a physical factor modifi es biochemical and physiological processes and infl uences enzyme systems improving the germination, growth and crop performance of plants (Dobrowolski et al. 1987, Hernandez et al. 2010, Szajsner 2009). In the pot growth experiment the germination capacity and rate were signifi cantly higher in the “Wiry” ecotype (Fig. 1a). Seed germinative capacity underwent a signifi cant increase after fi veand nine-fold semi-conductive laser irradiation, by, respectively, 13.3 and 14.2% as compared with the control seeds (Fig. 1b). The interaction of ecotypes with provided radiation dosages led to the conclusion that for the “Gajków” ecotype the D9 dosage provided a 16% capacity increase. The “Wiry” ecotype showed stimulation under the infl uence of D5 dosage – 16% increase as compared The improving infl uence of laser stimulation on phytoremediation capabilities of selected Silene vulgaris ecotypes 81 to the control group (Fig. 2). The germination rate underwent no signifi cant change under laser irradiation (Fig. 1b), but there was an interaction between ecotypes and dosages. The “Gajków” ecotype showed stimulation after D3, D7 and D9 seed irradiation (10% to over 13%), whereas the “Wiry” ecotype responded to D3 and D5 by 13% to 15% (Fig. 3). In the case of morphological features there were signifi cant differences between stem lengths among ecotypes, dosages and factor interactions. The above-ground parts of the “Gajków” ecotype (1.91 cm) were shorter than in the ‘Wiry’ ecotype (2.43 cm) – Fig. 4a. From among the radiation doses, D5 showed signifi cant lengthening of the stem (Fig. 4b). The obtained interaction showed a strong stimulating effect of seedbed irradiation with dosages D5 and D9 as compared to the control stems of the “Wiry” ecotype. For the “Gajków” ecotype, no signifi cant infl uence of laser irradiation was noted (Fig. 5). Leaf blade width showed no changes in either ecotype after pre-sowing irradiation of seed (Fig. 6). Table 1. Characteristics of the soil material (degraded) in conducted experiment Element Mobile form (Total form) [mg·kg-1] Element Mobile form [mg·100g-1] Ni 273.4 (1765.9) Mg 80.3 Cr 6.4 (373.1) P 0.8 Co 24.9 (153.1) K 2.9 Zn 97.0 (350.6) Element Volue (1M KCl)