Cd and Cr Uptake by Six Genotypes of Silene Vulgaris (Moench) Garcke A

PHYTOREMEDIATION Cd and Cr Uptake by Six Genotypes of Silene Vulgaris (Moench) Garcke A. Pradas de Real 1, R. Alarcón 2, P. García 3, M.M. Gil-Díaz 4, M.C. Lobo 5, A. Pérez-Sanz 6 1Department of Agroenvironmental Research, IMIDRA, 28800 Alcalá de Henares, SPAIN, [email protected] 2Department of Agroenvironmental Research, IMIDRA, 28800 Alcalá de Henares, SPAIN, [email protected] 3Department of Agroenvironmental Research, IMIDRA, 28800, Alcalá de Henares, SPAIN, [email protected] 4Department of Microbiology and Molecular Biology IV Faculty of Veterinary, UCM, 28040 Madrid SPAIN, [email protected] 5Department of Agroenvironmental Research, IMIDRA, 28800 Alcalá de Henares, SPAIN, [email protected] 6Department of Agroenvironmental Research, IMIDRA, 28800 Alcalá de Henares, SPAIN, [email protected] Abstract Cadmium and chromium are heavy metals released into the environment as a result of their use in large-scale industry. New research into technologies to remove these metals from polluted areas has become a major area of concern. Silene vulgaris (Moench) is a pioneer and facultative methallophyte. This species has multiple populations that have developed high levels of tolerance to heavy metals which makes it very interesting at the initial stages of revegetation and site decontamination. The objective of this work was to study the influence of S. vulgaris genotypes on Cd and Cr uptake. A semihydroponic experiment was carried out with cuttings of six genotypes of S. vulgaris (four hermaphrodites and two females) from different populations of Madrid (Spain). Cuttings were rooted and cultured in a phytotron chamber in black polyethylene trays filled with perlite and Hoagland nutrient solution. Plants were treated for 15 days as follows: (a) Control, nor Cd or Cr addition in nutrient solution (b) Cd 6 µM as CdSO 4 8H 2O (c) Cd 30 µM as CdSO 4 8H 2O (d) Cr (VI) 60 µM as K 2Cr 2O7 and (f) Cr (III) 60 µM as Clr 3Cr. Aerial part and root were dried and acid digested to measure total content of Cd and Cr by AA. Silene vulgaris showed genetically variability to uptake both metals even at genotypic level, and especially between hermaphrodite and female genotypes. The last one seems to be less efficient than hermaphrodite ones to metal uptake Key words: Cadmium, Chromium, Silene vulgaris , genotypes, metal uptake Introduction possesses multiple populations that have Cadmium and chromium are heavy evolved high tolerance levels to heavy met- metals used on a large scale in many differ- als. These characteristics made this species ent industries including metallurgical, pro- very interesting at the initial stages of reveg- duction of paints and pigments and tanning. etation and soil remediation. It exhibits high Additionally, Cd is generated in battery level of genetic variability, different life manufacturing and agriculture. In mam- forms and reproductive systems. Some pop- mals, Cd causes disorders in calcium and ulations of S. vulgaris present significant vitamin D metabolism, and it causes bio- high levels of metal tolerance. This alloga- mass reduction and chlorosis in plants. In mous species show a genetic variability the case of Cr, its impact in biological sys- regarding metal tolerance which is observed tems depends on metal speciation, which is within and between natural populations. responsible of its mobility, bioavailability Many populations have been proved to be and toxicity. The two most common and tolerant to heavy metal and their combina- stable chemical species of chromium in the tions, especially Cd, Zn and Pb. Data from environment are Cr(III) and Cr(VI). While these studies make S. vulgaris to be consid- Cr(III) is stable and non toxic, Cr(VI) causes ered an interesting species in the early stages oxidative stress and DNA damage in ani- of revegetation and soil restoration. Howev- mals and plants. er no information is available about its S. vulgaris (Moench) is a gynodioe- capacity to tolerate and accumulate chromi- cious, and facultative methallophyte. It is um. In order to avoid some problems associ- widely distributed throughout Europe and ated with high level of genetic variability 15 th ICHMET 563 PHYTOREMEDIATION within and between natural populations of S. rated, washed with distilled water and dried vulgaris , cuttings from selected genotypes in a forced air oven for 48 hours at 70 oC. Dry were used. Differences in the uptake and dis- weights were determined and plant material tribution of heavy metals among genotypes was grounded. Plant dried samples were of S. vulgaris have been reported only for Cu digested in an Anton Paar Microwave Reac- (Price and Abrahams 1994). The purpose of tion System 3000 by adding of 6ml of HNO 3 this work was to study the variability in 65% and 2ml H 2O2 33%. Afterwards, vol- metal uptake of Cd(II), Cr(III) and Cr(VI) of umes were brought up to 25ml with MQ six genotypes from Madrid Region (Spain). water and total concentrations of Cd and Cr were measured in the extracts with ICP and Materials and Methods Flame- AA. Six genotypes of S. vulgaris (Moench) Data were analyzed with a General were chosen from different populations of Lineal Model using the statistical package Madrid (Spain): Cadalso de los Vidrios (SV- SPSS version 16.0. The analysis were fol- 19), Rozas de Puerto Real (SV-21), Pinilla del lowed by a Duncan's multiple range test Valle (SV-27), Orusco (SV-30), Brea de Tajo (MRT) with a significant threshold for (SV-36), Valdemaqueda (SV-38). Genotypes p<0.05. Values in the tables and figures SV-19 and SV-38 were female, meanwhile indicate mean values. Significant differences the others were hermaphrodites. Cuttings among genotypes and treatments at p<0.05 were collected from field in "El Encín" are indicated by different letters. (Madrid, Spain) where they have been propa- gated vegetatively in plots of 1m 2. Just after Results and Discussion collecting, genotype cuttings were dipped Total cadmium concentrations are into rooting hormone Inavarplanté 1(ác. 3- shown in Table 1. In aerial part and roots, ondobutílico (A,B) 0.1%, ác. 1-nactilacético the higher the dose is applied, the greater (ANA) 0.1%, ziram 4%). Then they were absorption is observed in all genotypes. transferred into black polyethylene trays Therefore, the concentration of Cd in the tis- (five cuttings in each tray) with 40 sockets sues of S. vulgaris depends on the concentra- filled with vermiculite. Trays were filled in tion of Cd in the nutrient solution as previ- 3L of the corresponding solution and trans- ously reported by other authors (Chardon- ferred into a phytotron chamber (photoperi- nens et al. 1998). Differences are significant od 16h/8h, temperature 20 oC/16 oC, 165 also among genotypes. Female genotypes µmol photons m -2 s-1 ). Cuttings were root- show significant lower Cd uptake than her- ing with tap water for 2 weeks. Then they maphrodites. Among them, SV-21 presents were kept 1 week in Hoagland Nutrient solu- the highest Cd uptake in aerial part at high tion diluted 1/3, 1 week diluted 1/2 and Cd dosage. Hyperaccumulation of heavy another 3 weeks in full strength Hoagland metals depends on an interrelated network Nutrient solution: 3mM KNO 3, 2mM of physiological and molecular mechanisms Na(NO3)24H2O, 1mM NH4H2PO4, genetically controlled. Previous studies have 0.5mM MgSO47H2O, 50 µM NaCl, 25 µM found differences in the uptake and distribu- H3BO 3, 2 µM ZnSO 47H 2O, 2 µM tion of Cd among genotypes of soybean MnSO 4H 2O, 0.1 µM CuSO 45H 2O, (Arao et al. 2003), pea (Belimov et al. 2003) 0.5 µM (NH 4)6Mo 7O244H 2O, 20 µM Fe- and rice (Cheng et al. 2008). EDDHA. After this period of aclimatation Total Cr contents in aerial part and plants were randomly selected to be treated roots per plant are shown in table 2. There as follows: (a) Control, nor Cd or Cr addition are no significant differences between geno- in nutrient solution (b) Cd 6 µM as types to Cr uptake, but the trend shows a CdSO 48H 2O (c) Cd 30 µM as CdSO 48H 2O decrease in Cr uptake to female individuals. (d) Cr(VI) 60 µM as K 2Cr 2O7 and (f) Cr(III) Hermaphrodite SV-21 accumulates the 60 µM as Cl 3Cr. Four trays were used as greatest amount of Cr in aerial part and independent replicate of each treatment. roots. Differences among genotypes have The pH of the solutions was to 5.5 with been found in Indian mustard (Diwan et al. KOH before using. Nutrient solutions were 2008) and rice (Zeng et al. 2010) but in these replenished daily and completely changed studies, plants were treated with higher every 3 days. dosages than used in our experiment. Plants were harvested 2 weeks after treatment. Roots and aerial parts were sepa- 564 15 th ICHMET PHYTOREMEDIATION Total Cr uptake in S. vulgaris growth in the cell wall (Skeffington et al. 1976). nutrient solution with Cr(VI) was higher Chromium tends to be accumulated in roots than those treated with Cr(III). In some and poorly translocated into the aerial part cases, differences reach twice in aerial part as has been reported by (Shanker et al. 2005). and ten times in roots. The influence of spe- Despite being treated with two fold ciation on Cr uptake has been already report- more Cr than Cd, all genotypes accumulate ed in other species (Zayed et al. 1998, similar amounts of both metals in roots, but Gardea-Torresdey et al. 2005). This fact has the Cd concentration in aerial part tissue been explained based on Cr(VI) that is taken was 10% and 20% higher than Cr.

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