Prosopis laevigata a potential chromium (VI) and cadmium (II) hyperaccumulator desert plant

Bioresource Technology 101 (2010) 5862–5867

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Prosopis laevigata a potential chromium (VI) and cadmium (II) hyperaccumulator desert plant L. Buendía-González a,b,*, J. Orozco-Villafuerte b, F. Cruz-Sosa a, C.E. Barrera-Díaz b, E.J. Vernon-Carter c a

Departamento de Biotecnología, Universidad Autónoma Metropolitana-Iztapalapa, Av. San Rafael Atlixco, No. 186, Col. Vicentina, Iztapalapa, D.F. México C.P. 09340, Mexico Facultad de Química, Universidad Autónoma del Estado de México, Paseo Colón esq. Paseo Tollocan s/n, Col. Residencial Colón, Toluca, Estado de México C.P. 50120, Mexico c Departamento de Ingeniería de Procesos e Hidráulica, Universidad Autónoma Metropolitana-Iztapalapa, Av. San Rafael Atlixco, No. 186, Col. Vicentina, Iztapalapa, D.F. México C.P. 09340, Mexico b

a r t i c l e

i n f o

Article history: Received 23 March 2009 Received in revised form 4 March 2010 Accepted 4 March 2010 Available online 29 March 2010 Keywords: Prosopis laevigata Hyperaccumulator Translocation factor In vitro culture Phytoremediation

a b s t r a c t The bioaccumulation of Cr(VI) and Cd(II) in Prosopis laevigata and the effect of these heavy metals on plant growth were assessed. P. laevigata seeds were cultured during 50 days on modified Murashige–Skoog medium supplemented with four different concentrations of Cr(VI) (0–3.4 mM) and Cd(II) (0–2.2 mM), respectively. Heavy metals did not stop germination, but smaller plants with fewer leaves and secondary roots were produced. Seedlings showed an accumulation of 8176 and 21,437 mg Cd kg1 and of 5461 and 8090 mg Cr kg1 dry weight, in shoot and root, when cultured with 0.65 mM Cd(II) and 3.4 mM Cr(VI), respectively. These results indicated that significant translocation from the roots unto aerial parts took place. A bioaccumulation factor greater than 100 for Cd and 24 for Cr was exhibited by the seedlings. P. laevigata can be considered as a potential hyperaccumulator of Cd(II) and Cr(VI) species and considered as a promising candidate for phytoremediation purposes. Ó 2010 Elsevier Ltd. All rights reserved.

1. Introduction One of the most serious problems facing the world today is the environmental contamination caused by industrial wastes. Industry and mining activities produce innumerable toxic substances such as pesticides, organ-chlorides, greases and oils, heavy metals, toxins and other aggressive chemical elements which are directly discharged to sea, rivers, lagoons and lands. Cadmium (II) is a toxic heavy metal associated with Zn mining and industrial operations where cadmium has been used to prevent corrosion of machinery. Resulting air-borne cadmium dust represents a significant health hazard. Moreover, the main anthropogenic sources of Cd (mining, smelting and metal finishing) often contain others metals, such as Cr, Pb, Ni, Se, As, and Zn which frequently pollute soil and water (Adriano, 2001). The Environmental Protection Agency (EPA) of the United States has classified cadmium as a Group B1 contaminant, meaning that it is a probable human carcinogen, when exceeding a permissible limit in water of 5 ppb (Peer et al., 2005). Chromium

* Corresponding author at: Departamento de Biotecnología, Universidad Autónoma Metropolitana-Iztapalapa, Av. San Rafael Atlixco No. 186, Col. Vicentina, Iztapalapa, D.F. México C.P. 09340, Mexico. Tel.: +52 55 5804 4714; fax: +52 55 5804 4712. E-mail addresses: [email protected] (L. Buendía-González), jov202001@ yahoo.com.mx (J. Orozco-Villafuerte), [email protected] (F. Cruz-Sosa), [email protected] (C.E. Barrera-Díaz), [email protected] (E.J. Vernon-Carter). 0960-8524/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2010.03.027

is present in many oxidation states in the environment, including the most common forms Cr(0), Cr(III), and Cr(VI). Cr(III) is an essential nutrient for animals, and Cr(VI) is considered to be 1000 times more toxic than Cr(III) (Peer et al., 2005). The World Health Organization has determined that Cr(VI) is a human carcinogen, and EPA has classified it as a Group A contaminant, that is, a human carcinogen by the inhalation route of exposure, when exceeding a permissible limit for total chromium in water of 100 ppb (Peer et al., 2005). Toxic metal contamination of soil, aqueous waste streams and groundwater poses a major environmental and human health problem which is still in need of an effective and affordable technological solution. In attempting to preserve our environment, new methods of remediation using physical, chemical and biological principles are being studied (Cunningham and Berti, 1993). Remediation of metal-contaminated soil faces a particular challenge, because unlike organic contaminants, metals cannot be degraded, and they must be removed. Phytoremediation is defined as the use of plants to remove, contain, or render harmless environmental contaminants; this includes the use of vegetation for in situ treatment of water, sediments, soils, and air. Phytoextraction is an aspect of phytoremediation that involves the removal of toxins, especially heavy metals and metalloids, by the roots of the plants with subsequent transport to aerial plant organs (Salt et al., 1998). For considering phytoextraction as a feasible remediation tool, plants must be able to take up large concentrations of

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heavy metals into the roots, to translocate these metals to the aerial parts (shoots) so large concentrations are accumulated in them, and to produce large biomass (Cunningham et al., 1995). The use of plants to extract toxic metals from contaminated soils has emerged as a cost-effective and environment-friendly cleanup alternative. Assays in vitro conditions allow us to select potential plants for phytoremediation purposes, and also for distinguishing unequivocally between the plant activity responses and those derived from native microorganisms existing in soils (Reynoso-Cuevas et al., 2008). Helianthus annuus is a species that has been used for phytoremediation studies in soils contaminated with heavy metals (Kališová-Špirochová et al., 2003; Meers et al., 2005; De la Rosa et al., 2008). However, this species is only suitable for use in temperate and tropical climates. In Mexico, many heavy metals contaminated sites are predominantly situated in semi-arid and arid regions. Desert plants have adapted to tolerate and thrive in harsh desert conditions such as extreme temperatures, high salt content and nutrient deficiency in soils. Because of their highly specialized physiologies, desert plants such as Prosopis laevigata, are obvious candidates for examination of other stress-induced adaptive mechanisms such as growth in heavy metal-contaminated sites (Aldrich et al., 2003), as demonstrated in studies in genus Prosopis (Armienta et al., 2008; Haque et al., 2009; Buendía-González et al., 2010). The aim of this work was to investigate the in vitro ability of P. laevigata (mesquite), a widely distributed species in the semi-arid and arid regions in Mexico, to remove two different heavy metals in different concentrations from the culture media, and to assess the effect of these metals uptake on the growth, morphology and survival of the plant. 2. Methods 2.1. Plant material Mature brown pods were collected from adult P. laevigata (Humb. and Bonpl. ex Willd M.C. Johnston) trees growing naturally in the Mexican State of San Luis Potosi. Mature seeds were isolated from the pods and were scarified mechanically, under laminar flow hood, disinfested by immersing them in ethanol, followed by immersion in sodium hypochlorite (Buendía-González et al., 2007). Seeds were carefully rinsed with sterile bi-distilled water five times and germinated aseptically in culture tubes (25  150 mm) containing 15 mL of liquid modified Murashige and Skoog medium (MS) (Murashige and Skoog, 1962). Filter paper (10.1 cm  1.5 cm, Whatman 1004 917) segments were put inside the culture tubes for the purpose of supporting the seeds. One seed was aseptically placed into each culture tube and a set of five culture tubes was evaluated for each treatment. 2.2. Heavy metals tested The heavy metal (HM) sources used in this study included K2Cr2O7 and CdCl22½H2O salts (Baker Analyzed, Phillipsburg NJ). Stock solutions of each heavy metal salt were prepared at a concentration of 1000 mg L1. 2.3. Media preparation and culture conditions The modified MS medium was prepared as follows: (1) some salts contained in the original MS medium are prone to react violently or form precipitates when entering in contact with the heavy metals. Thus the medium only contained the following concentrations of macroelements: 1.65 g L1 NH4NO3, 0.37 g L1 MgSO47H2O and 0.44 g L1 CaCl22H2O, and of microelements:

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6.2 mg L1 H3BO3, 22.30 mg L1 MnSO44H2O, 0.25 mg L1 Na2MoO42H2O 0.025 mg L1 CuSO45H2O, 0.025 mg L1 CoCl26H2O, respectively; (2) 10 g L1 sucrose was added; and (3) aliquots of the heavy metal salt stock solutions were added in order to achieve HM concentrations of 0.0, 0.5, 1.0, 2.0, 3.4 mM of Cr(VI) and 0.0, 0.3, 0.65, 1.3, 2.2 mM of Cd(II) in the liquid culture media. All media were adjusted to pH 5.8 with 1 N NaOH and 1 N HCl before autoclaving at 121 °C for 18 min. All cultures were maintained at 25° ± 2 °C under warm-white fluorescent light at an irradiance of 50 mol m2 s1 and a 16 h (light) 8 h (dark) photoperiod. Cr(VI) was quantified to ensure that its concentration was the same before and after the autoclaving process in all the treatments, by reacting it with 1,5-diphenylcarbazide in acid solution reagent (APHA, 1992). Measurements were made in a spectrophotometer (Cecil 3000 Series) at 540 nm.

2.4. Evaluation of plant growth and heavy metal resistance One seed was aseptically placed into each culture tube. Percentage of seed germination was recorded after 10 days of culture. Morphological observations and growth seedling measurements were made at 10 day intervals during 50 days. The root and shoot lengths of the seedlings inside the culture sealed tubes were measured. Seedling length was measured from the main root apex to the main shoot apex. Surviving seedlings were harvested after 50 days, rinsed with deionized water three times and dried in a convection oven at 60 °C for 72 h. Their dry weight was determined, and this value was considered as the plant biomass. The stem and root lengths, and dry weight measurements were determined for each seedling. Every treatment consisted of a batch of five seedlings. Means derived from three batches were used for statistical analysis (n = 3). Growth measurements were used for evaluating the growth ratio (GR) and the heavy metal tolerance index (TI) resistance indicators, which are defined as (Baker, 1987):

plant biomass HM  100 plant biomass without HM ðcontrolÞ root length HM TI ¼ root length without HM ðcontrolÞ

GR ¼

ð1Þ ð2Þ

2.5. Analysis of cadmium and chromium contents in biomass Fifty day-old surviving seedlings were separated into root and aerial parts and were dried as indicated in the previous section. Dried tissue was weighed, powdered and digested with 5 mL of concentrated HNO3 in a microwave oven (CEM Mars5, CEM corporation, Mathews, North Carolina), and the final sample volume was adjusted to 10 mL with deionized water and placed in HDPE flasks. The metals concentration was analyzed from those samples using a Varian Spectra AA-220 FS Atomic Absorption Spectrometer (Varian, Australia). The concentration of Cd and Cr were determined by calibration curves obtained using standards solutions of pure metal ions (Baker Analyzed, Phillipsburg, NJ). The standard calibration curves had correlation coefficients (r2) of 0.99 or better. All glassware and apparatus were washed with 0.1 N HNO3 before use. The shoot and root heavy metal contents were determined for each seedling. Every treatment consisted of a batch of five seedlings. Means derived from three batches were used for statistical analysis (n = 3). Metal concentration measurements were used for evaluating the bioaccumulation factor (BF) and translocation factor (TF), which are defined as (Zhao et al., 2003; Niu et al., 2007):

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½HM in shoot ½HM in culture ½HM in shoot TF  ½HM in root BF 

8

ð3Þ ð4Þ length (cm)

6

2.6. Statistical analysis All the experiments were done in quintuplicate with three replicates. All the experimental data obtained in this work were subjected to an analysis of variance (ANOVA) using the NCSS version five statistical software (Wireframe Graphics, Kaysville, UT) (NCSS 2001). Comparisons of means were made using Tukey’s multiple range test at the 5% level of probability for all experiments.

4

2

0 0

1

2

3

4

HM concentration (mM) Fig. 2. Shoot and root length of Prosopis laevigata after 50 days of culture in media enriched with heavy metals. (a) Root length with Cr(VI) ( j ); (b) root length with Cd(II) ( N ); (c) shoot length with Cr(VI) (– j –); and (d) shoot length with Cd(II) (– N –). Vertical bars denote SD.

3. Results 3.1. Heavy metals effect on germination, survival response, and plant growth P. laevigata seeds showed germination of 100% irrespective of the Cd(II) and Cr(VI) concentrations used, but the morphologic characteristics of the seedlings were affected by both chromium and cadmium, manifested in a smaller size, lower number of leaves, and lower number of secondary roots in the seedlings compared to the control treatment without HM. Both, the HM and control treatments germinated after 24 h, so that germination time was not affected by HM. Cadmium had a more pronounced effect on these seedling characteristics than chromium. The survival response of P. laevigata evaluated as the percentage of surviving plants from five seeds planted, indicated that the Cd(II)-treated plants (0.3, 0.65, 1.3, and 2.2 mM) showed a significantly earlier death time (30 days) and significantly lower survival percentages than the Cr(VI)-treated plants (0.5, 1.0, 2.0 and 3.4 mM) (50 days) (p < 0.05) (Fig. 1). Furthermore, cadmium at a concentration of 1.3 and 2.2 mM caused the death of most seedlings by the end of the fourth week. The effects of Cd(II) and Cr(VI) concentrations at 50 days, on shoot and root length in P. laevigata plants are shown in Fig. 2. In general terms, data showed a reduction in shoot and root elongation as HM concentrations increased. The shoot size of the plants exposed to 0.3, 0.65, 1.3, and 2.2 mM Cd(II) was reduced by 28.37%, 44.68%, 56.03% and 75.46%, and those exposed to 0.5, 1.0, 2.0, and 3.4 mM Cr(VI) showed a shoot size reduction of 3.55%, 15.60%, 22.70% and 34.04%, respectively, when compared to the control shoot elongation. The root elongation of the plants exposed to 0.3, 0.65, 1.3, and 2.2 mM Cd(II) was reduced by 78.9%, 88.5%, 91.7% and 93.1%, while those exposed to 0.5, 1.0, 2.0, and 3.4 mM

Cr(VI) showed reductions of 46.8%, 65.6%, 63.3% and 61.5%, respectively, compared to the control root size. Regarding the resistance of P. laevigata seedlings, it was observed lower tolerance index (TI) values in Cd(II)-treated plants than in the Cr(VI)-treated plants (Fig. 3). The TI was significantly greater for the plants treated with the lowest Cd(II) (0.3 mM) or Cr(VI) (0.5 mM) concentrations. Another way of measuring the resistance of seedlings to heavy metals is by determining the growth ratio (GR). The GR of P. laevigata was evaluated at two concentrations of Cd(II) (0.3 and 0.65 mM) and of Cr(VI) (2.0 and 3.4 mM). Whereas the GR decreased as Cd(II) concentration increased, the opposite behavior occurred with Cr(VI) (Fig. 4). Mesquite produced significantly higher dry biomass at 3.4 mM Cr(VI) and 0.3 mM Cd(II) than at 2.0 mM Cr(VI) and 0.65 mM Cd(II), respectively. An increase from 0.3 to 0.65 mM Cd(II) produced a dry weight reduction in the seedlings from 38.15% to 48.10%, whereas the growth of plants in medium containing chromium showed a reduction in dry weight of 50.47% and 25.35% for 2.0 and 3.4 mM Cr(VI), respectively, with regard to the control. 3.2. Heavy metal uptake The Cd(II)-treated plants accumulated 9404 and 21,437 mg of Cd kg1 of root dry tissue at 0.3 and 0.65 mM Cd(II), whereas the roots of Cr(VI)-treated plants accumulated 5035 and 8090 mg of Cr kg1 at 2.0 and 3.4 mM Cr(VI), respectively. The shoot tissue of Cd(II)-treated plants accumulated 3811 and 8176 mg Cd kg1 of dry tissue at 0.3 and 0.65 mM Cd(II), whereas the shoots of

1 0.9

HM tolerance index

survival response (%)

100 80 60 40 20

0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0 0

0 0

1

2

3

4

0.5

1

1.5

2

2.5

3

3.5

HM concentration (mM)

HM concentration (mM) Fig. 1. Survival response for Prosopis laevigata at the end of 50 days of culture in media supplemented with Cr(VI) (– j ) and Cd(II) ( N ) heavy metals. Vertical bars denote SD (n = 3).

Fig. 3. Effect of various concentrations of heavy metals on tolerance index (root length HM/root length without HM) of Prosopis laevigata after 50 days of culture in media enriched with heavy metals. Treatments with Cr(VI) (– j –); and Cd(II) (– N –). Vertical bars denote SD.

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140

Bioaccumulation factor

120

Growth ratio (%)

80

60

40

20

100 80 60 40 20 0

0

Cd 0.3

Cd 0.65

Cr 2.0

Cd 0,3

Cr 3.4

Fig. 4. Growth ratio (%) (plant biomass HM/plant biomass without HM) of Prosopis laevigata after 50 days of culture in media enriched with heavy metals. Vertical bars denote SD.

Cd 0,65

Cr 2,0

Cr 3,4

HM concentration (mM)

HM concentration (mM)

Fig. 6. Values of bioaccumulation factor (shoot/culture concentration ratio) of P. laevigata in Cd(II) and Cr(VI) treatments. Vertical bars denote SD.

0.8

4. Discussion The toxic effect of polluting agents on the germination of seeds is a good indicator of the possible tolerance of the plant against these contaminants (Carrillo-Castañeda et al., 2002), so that is important to study the effect of HM on seeds response. P. laevigata seeds used in this study showed germination of 100%. Although both metals showed a toxic effect on seedlings, cadmium had a more pronounced effect on these seedling characteristics than chromium. A study with alfalfa (Medicago sativa) using cadmium and chromium showed a reduction in seed germination at lower doses (10–40 mg l10.1–0.7 mM Cr(VI) and 0.09–0.35 mM Cd(II)) (Peralta et al., 2001) than those used in this study. Studies by Parr (1982) showed that at high levels of Cr(VI) in soil

Shoots

Roots

20000

15000

-1

mg HM kg dry weight

25000

10000

5000

0

Cd 0,3

Cd 0,65

Cr 2,0

Cr 3,4

HM concentration (mM) Fig. 5. Bioaccumulation of Cd and Cr by P. laevigata in in vitro cultures. Vertical bars denote SD.

0.7

Translocation factor

Cr(VI)-treated plants accumulated 2364 and 5461 mg Cr kg1 at 2.0 and 3.4 mM Cr(VI), respectively (Fig. 5). The heavy metal uptake at the two highest concentrations of cadmium was not examined because the seedlings were dead at harvesting time. P. laevigata showed a BF for Cd(II) above 100, independent of Cd concentration in the medium (Fig. 6). In the case of the Cr(VI)-treated plants, the bioaccumulation factor was slightly higher than 24, also independent of Cr concentration in the medium. The Cd(II)treated plants showed a TF of 0.38 and 0.41 for 0.3 and 0.65 mM Cd(II), whereas the Cr(VI)-treated plants showed TF of 0.52 and 0.67 for 2.0 and 3.4 mM Cr(VI), respectively (Fig. 7). These TF values showed that mesquite was more efficient for translocation of Cr than Cd, as a significant higher TF value was found for the 3.4 mM Cr(VI) treatment.

0.6 0.5 0.4 0.3 0.2 0.1 0

Cd 0.3

Cd 0.65

Cr 2.0

Cr 3.4

HM concentration (mM) Fig. 7. Values of translocation factor (shoot/root concentration ratio) of P. laevigata in Cd(II) and Cr(VI) treatments. Vertical bars denote SD.

(500 mg kg1) germination and growth of bush bean were highly affected. The survival response of P. laevigata indicated that the Cd(II)treated plants showed a significantly earlier death time and significantly lower survival percentages than the Cr(VI)-treated plants. These data demonstrated that the mesquite was more tolerant to Cr(VI) than to Cd(II) toxicity. A study with six different species (Cynodon dactylon, Pluchea indica, Phyllanthus reticulatus, Echinochloa colonum, Vetiveria nemoralis, and Amaranthus viridis) exposed to Cr(VI) concentrations of 50–400 ppm (0.9–7.6 mM) showed a reduction in percent survival of 89–0% (Sampanpanish et al., 2006). The effects of Cd(II) and Cr(VI) concentrations on shoot and root length in P. laevigata plants showed a reduction in shoot and root elongation as HM concentrations increased. Thus, the deleterious effect of HM was more pronounced on seedlings grown in medium supplied with Cd(II) than with Cr(VI). Öncel et al. (2000) reported that a reduced length in wheat occurred when exposed to Cd(II), and a diminution in the production of chlorophyll a and b. The ways of measuring the resistance of seedlings to heavy metals is by determining the tolerance index (TI) and the growth ratio (GR). The resistance of seedlings to heavy metals in this study showed that mesquite was more tolerant to chromium than to cadmium, as both measures of resistance were higher in the Cr(VI)treated plants. The significant higher toxic effect of cadmium in the mesquite seedlings can be due, besides the intrinsic toxicity of the heavy metal, to the fact that the culture medium was made from a salt source that did not contain potassium, which is essential for the plant metabolism. On the other hand, salt containing potassium was used for preparing the medium containing chromium. Deficiency of potassium results in loss of cell turgor, flaccid tissues and an increased susceptibility to drought, salinity, frost

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damage and fungal attack (George and De Klerk, 2008) in whole plants. In addition, to avoid toxicity, plants have also been documented to catalyze redox reactions and alter the chemistry of metal ions. Lytle et al. (1998) showed that from a solution supplemented with toxic Cr(VI), water hyacinth (Eichornia crassipes) accumulated nontoxic Cr(III) in roots and shoots. The Cr(VI) to Cr(III) reduction apparently took place in the fine lateral roots. Then, the reduced ion (Cr(III)) was subsequently translocated to leaf tissues. The Cd(II)-treated plants accumulated more metal in root and shoot tissue than the Cr(VI)-treated plants. The statistical analysis showed that the averages of Cd and Cr found in the root and shoot tissues were significantly different. These statistical results suggest that HM concentration tended to affect the Cr(VI) and Cd(II) uptake capacities of the mesquite seedlings. The uptake results from Cr(VI)-treated P. laevigata plants showed similar results with Prosopis spp. at 125 ppm of Cr(VI) (2.4 mM) in agar, which showed a Cr(VI) uptake of 10,983, 2262, and 991 mg Cr kg1 dry weight (dw) in root, stem and leaf, respectively (Aldrich et al., 2003). The uptake results from Cd(II)-treated P. laevigata plants showed similar or better results than with M. sativa and Thlaspi hyperaccumulators species (Peralta et al., 2001; Pongrac et al., 2009). M. sativa at 20 mg L1 (0.18 mM) of Cd(II) in agar showed a Cd(II) uptake of 6710 and 4145 mg kg1 dw in root and shoot (Peralta et al., 2001), whereas Thlaspi species at 50–250 mg kg1 of Cd(II) in peat-based substrate showed a Cd(II) uptake of 2500–3700 and 5000–5250 mg kg1 dw in root and shoot (Pongrac et al., 2009), respectively. Although P. laevigata accumulated more Cd than Cr, the results of this work clearly indicate that Cd(II) was more toxic than Cr(VI), causing more damage which resulted in a lower growth and survival of plants. This might have happened because cadmium is an analogue of zinc and iron (Peer et al., 2005), essential nutrients for plants, which were not added in the culture medium. There are few reports in the literature regarding species capable of accumulating 100 mg or more of Cd in the aerial plant tissues (Reeves and Baker, 2000). Species capable of accumulating metals at levels 100fold or higher in the shoots with regard to common non-accumulator plants, are considered as hyperaccumulators (Lasat, 2002). Ideally, these plants should accumulate metals at levels up to 0.1–1.0% of the dry weight plant biomass produced, depending on the specific metal being taken up (Pilon-Smits, 2005). Thus, a hyperaccumulator will concentrate more than 100 mg Cd kg1 dw, and more than 1000 mg Cr kg1 dw (Lasat, 2002). According to these results of HM accumulation, P. laevigata might be considered a potential hyperaccumulator species of both heavy metals. Metal concentrations in shoot tissues are generally the standard used for defining a hyperaccumulator but the accumulation factor is highly recommended for understanding the feasibility of phytoextraction. The bioaccumulation factor (BF) is more important than shoot concentration for considering the potential of phytoextraction for a given species. P. laevigata results showed a BF greater than 100 for Cd(II) and 24 for Cr(VI). In metal excluder species, the bioaccumulation factor is typically lower than 1, whereas in metal accumulator species the factor is often greater than 1 (Baker, 1981). Translocation factor (TF) values can describe the movement and distribution of heavy metals in plants. The Cd(II)-treated plants showed a TF above 0.38, whereas the Cr(VI)-treated plants showed TF above 0.52. Transport across root cellular membrane is an important process which initiates metal absorption into plant tissues. One study of heavy metals translocation into peanut fruits provided evidence that accumulation occurred predominantly via phloem (Popelka et al., 1996). For the phytoextraction process, substantial amounts of the HM must be removed by the root from the medium, and translocate them to the harvestable plant parts,

so that they can be completely removed from the contaminated site (Zayed and Terry, 2003). The results indicate that P. laevigata is a suitable candidate species for the removal of HM in large scale phytoremediation purposes, as it proved to be a hyperaccumulator of Cd(II) and Cr(VI), resist the toxic effects of these metals, and exhibited good bioconcentration factors. 5. Conclusions P. laevigata was able to germinate and grow in different Cd(II) and Cr(VI) concentrations. The growth ratio by the seedlings for both heavy metals was over 50%. Also, the seedlings had the capacity to uptake Cd and Cr, but their ability to accumulate these heavy metals was different. The deleterious effect of heavy metals on seedlings growth was more pronounced when the medium was supplied with Cd(II) than with Cr(VI). The Cd(II)-treated plants accumulated 9404 and 21,437 mg of Cd kg1 of root dry tissue at 0.3 and 0.65 mM Cd(II), and translocated 38% of the metal to the shoot tissues (3811 and 8176 mg Cd kg1 of dry tissue at 0.3 and 0.65 mM Cd(II)), respectively. The roots of Cr(VI)-treated plants accumulated 5035 and 8090 mg of Cr kg1 at 2.0 and 3.4 mM Cr(VI), and translocated 52% of the metal to the shoot tissues (2364 and 5461 mg Cr kg1 at 2.0 and 3.4 mM Cr(VI)), respectively. This work showed that P. laevigata is a potential hyperaccumulator species of Cd(II) and Cr(VI), and may be grown directly in soils highly contaminated with both HM, and thus is a promising prospect for heavy metals phytoremediation purposes occurring in arid and semi-arid climates. Acknowledgement The first author wishes to thank the Consejo Nacional de Ciencia y Tecnología (CONACyT) for her postdoctoral scholarship. References Adriano, D.C., 2001. Trace elements in terrestrial environments, second ed.. Biogeochemistry, Bioavailability and Risk of Metals Springer-Verlag, New York. p. 867. Aldrich, M.V., Gardea-Torresdey, J.L., Peralta-Videa, J.R., Parsons, J.G., 2003. Uptake and reduction of Cr(VI) to Cr(III) by mesquite (Prosopis spp.): chromate–plant interaction in hydroponic and solid media studied using XAS. Environmental Science and Technology 37 (9), 1859–1864. APHA, 1992. Standard Methods for the Examination of Water and Wastewater, 17 ed. American Public Health Association, Washington, DC. Armienta, M.A., Ongley, L.K., Rodríguez, R., Cruz, O., Mango, H., Villaseñor, G., 2008. Arsenic distribution in mesquite (Prosopis laevigata) and huizache (Acacia farnesiana) in the Zimapán mining area, México. Geochemistry: Exploration, Environment, Analysis 8 (2), 191–197. Baker, A.J.M., 1981. Accumulators and excluders-strategies in the response of plants to heavy metals. Journal of Plant Nutrition 3 (1), 643–654. Baker, A.J.M., 1987. Metal tolerance. New Phytologist 106 (s1), 93–111. Buendía-González, L., Orozco-Villafuerte, J., Cruz-Sosa, F., Chávez-Ávila, V.M., Vernon-Carter, E.J., 2007. Clonal propagation of mesquite tree (Prosopis laevigata Humb. and Bonpl. ex Willd. M.C. Johnston). I. Via cotyledonary nodes. In Vitro Cellular and Developmental Biology – Plant 43 (3), 260–266. Buendía-González, L., Orozco-Villafuerte, J., Cruz-Sosa, F., Estrada-Zúñiga, M.E., Barrera-Díaz, C.E., Vernon-Carter, E.J., 2010. In vitro lead and nickel accumulation in mesquite (Prosopis laevigata) seedlings. Revista Mexicana de Ingeniería Química 9 (1), 1–9. Carrillo-Castañeda, G., Juárez Muños, J., Peralta-Videa, J.R., Gomez, E., DuarteGardea, M., Tiemann, K.J., Gardea-Torresdey, J.L., 2002. Alfalfa growth promotion by bacteria growth under iron limiting conditions. Advances in Environmental Research 6 (3), 391–399. Cunningham, S.D., Berti, W.R., 1993. Remediation of contaminated soils with green plants: an overview. In Vitro Cellular and Developmental Biology – Plant 29 (4), 207–212. Cunningham, S.D., Berti, W.R., Huang, J.W., 1995. Phytoremediation of contaminated soils. Trends in Biotechnology 13 (9), 393–397. De la Rosa, G., Cruz-Jiménez, G., Cano-Rodríguez, I., Fuentes-Ramírez, R., GardeaTorresdey, J.L., 2008. Effect of the plant stage and presence of a chelating agent on the tolerance and absorption of Cr(III) by Hellianthus annuus. Revista Mexicana de Ingeniería Química 7 (3), 243–257. George, E.F., De Klerk, G.J., 2008. The components of plant tissue culture media I: macro- and micro-nutrients. In: George, E.F., De Klerk, G.J., Hall, M.A. (Eds.),

L. Buendía-González et al. / Bioresource Technology 101 (2010) 5862–5867 Plant Propagation by Tissue Culture, third ed. Springer, Dordrecht, The Netherlands, pp. 65–113. Haque, N., Peralta-Videa, J.R., Duarte-Gardea, M., Gardea-Torresdey, J.L., 2009. Differential effect of metals/metalloids on the growth and element uptake of mesquite plants obtained from plants grown at a copper mine tailing and commercial seeds. Bioresource Technology 100, 6177–6182. Kališová -Špirochová, I., Puncˇochárˇová, J., Kafka, Z., Kubal, M., Soudek, P., Vaneˇk, T., 2003. Accumulation of heavy metals by in vitro cultures of plants. Water, Air, and Soil Pollution: Focus 3 (3), 269–276. Lasat, M.M., 2002. Phytoextraction of toxic metals: a review of biological mechanisms. Journal of Environmental Quality 31 (1), 109–120. Lytle, C.M., Lytle, F.W., Yang, N., Qian, J.H., Hansen, D., Zayed, A., Terry, N., 1998. Reduction of Cr(VI) to Cr(III) by wetland plants: potential for in situ heavy metal detoxification. Environmental Science and Technology 32, 3087–3093. Meers, E., Ruttens, A., Hopgood, M.J., Samson, D., Tack, F.M.G., 2005. Comparison of EDTA and EDDS as potential soil amendments for enhanced phytoextraction of heavy metals. Chemosphere 58 (8), 1011–1022. Murashige, T., Skoog, F., 1962. A revised medium for rapid growth and bioassay with tobacco tissue cultures. Physiologia Plantarum 15 (3), 473–497. NCSS, 2001. NCSS – Number Cruncher Statistical Software. Kaysville, Utah, USA. Niu, Z.X., Sun, L.N., Sun, T.H., Li, Y.S., Wang, H., 2007. Evaluation of phytoextracting cadmium and lead by sunflower, ricinus, alfalfa and mustard in hydroponic culture. Journal of Environmental Sciences 19 (8), 961–967. Öncel, I., Kele, Y., Üstün, A.S., 2000. Interactive effects of temperature and heavy metal stress on the growth and some biochemical compound in wheat seedlings. Environmental Pollution 107 (3), 315–320. Parr, P.D., 1982. Publication No. 1761. Environmental Sciences Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee. Peer, W.A., Baxter, I.R., Richards, E.L., Freeman, J.L., Murphy, A.S., 2005. Phytoremediation and hyperaccumulator plants. In: Tamás, M.J., Martinoia, E.

5867

(Eds.), Topics in Current Genetics: Molecular Biology of Metal Homeostasis and Detoxification, vol. 14. Springer-Verlag, Berlin Heidelberg, pp. 299–340. Peralta, J.R., Gardea-Torresdey, J.L., Tiemann, K.J., Gomez, E., Arteaga, S., Rascon, E., Parsons, J.G., 2001. Uptake and effects of five heavy metals on seed germination and plant growth in alfalfa (Medicago sativa L.). Bulletin of Environmental Contamination and Toxicology 66 (6), 727–734. Pilon-Smits, E., 2005. Phytoremediation. Annual Review of Plant Biology 56, 15–39. Pongrac, P., Fang-Jie, Z., Razinger, J., Zrimec, A., Regvag, M., 2009. Physiological responses to Cd and Zinc in two Cd/Zn hyperaccumulating Thlaspi species. Environmental and Experimental Botany 66, 479–486. Popelka, J.C., Schubert, S., Schulz, R., Hansen, A.P., 1996. Cadmium uptake and translocation during reproductive development of peanut (Arachis hypogaea L.). Angewandte Botanik 70, 140–143. Reeves, R.D., Baker, A.J.M., 2000. Metal-accumulation plants. In: Raskin, I., Ensley, B.D. (Eds.), Phytoremediation Toxic Metals: Using Plants Clean Up Environment. John Wiley and Sons, New York, pp. 193–229. Reynoso-Cuevas, L., Gallegos-Martínez, M.E., Cruz-Sosa, F., Gutiérrez-Rojas, M., 2008. In vitro evaluation of germination and growth of five plant species on medium supplemented with hydrocarbons associated with contaminated soils. Bioresource Technology 99 (14), 6379–6385. Salt, D.E., Smith, R.D., Raskin, I., 1998. Phytoremediation. Annual Review of Plant Physiology and Plant Molecular Biology 49, 643–668. Sampanpanish, P., Pongapich, W., Khaodhiar, S., Khan, E., 2006. Chromium removal from soil by phytoremediation with weed plant species in Thailand. Water, Air and Soil Pollution 6 (1–2), 191–206. Zayed, A.D., Terry, N., 2003. Chromium in the environment: factors affecting biological remediation. Plant and Soil 249 (1), 139–156. Zhao, F.J., Lombi, E., McGrath, S.P., 2003. Assessing the potential for zinc and cadmium phytoremediation with the hyperaccumulator Thlaspi caerulescens. Plant and Soil 249 (1), 37–43.