Germination and sporophytic development of Regnellidium diphyllum Lindm . ( Marsileaceae ) in the presence of copper

Regnellidium diphyllum Lindm. is a heterosporous fern growing in wetlands and humid soils that are being converted to agricultural activities. Many products that are used in agriculture contain copper, resulting in surface and groundwater contamination. Germination and initial development tests were performed using Meyer’s solution containing copper sulphate at concentrations of 0 (control), 1, 5, 10, 50 and 100 mg L−1. Th e experiment was conducted in a growth chamber at 25 ± 1°C for 28 days, with a 12/12-hour light/dark cycle and a photon fl ux density of 100 μmol m−2 s−1. Th e lowest germination rate (6%) was observed at 100 mg L−1. Primary root growth was signifi cantly reduced at ≥ 10 mg L−1. Secondary leaves of sporophytes grown in concentrations ≥ 5 mg L−1 were progressively shorter than were those formed by the control plants. We conclude that the release of pollutants containing copper into the natural habitats of R. diphyllum can cause phytotoxicity, threatening the establishment of populations and worsening the already vulnerable conservation status of this species.


Introduction
Aquatic ecosystems can be contaminated by heavy metals as a result of natural processes, such as the weathering of rocks, as well as by anthropogenic infl uences such as industrial activities, agricultural activities and domestic effl uents (Soares et al. 2000;Hu et al. 2010).Because of its widespread use in pesticides and because it is a byproduct of many industrial activities, copper is considered to be a major pollutant (Mal et al. 2002).According to the Brazilian National Environmental Council, the current regulatory limit for copper in water is 0.009 mg L −1 (Brasil 2005).
As one of the prosthetic groups of enzyme systems and a facultative activator of enzyme systems (Baker 1990), copper is considered to be an essential micronutrient for plants (Arnon & Stout 1939).However, when present in excess, it can be phytotoxic, causing disorders of growth and development by adversely aff ecting important physiological processes (Yruela 2005).Th e consequences of copper toxicity are generally more severe for plants in aquatic ecosystems, because they can absorb the metal via roots and leaves, rather than via the roots alone (Guilizzoni 1991).
In the state of Rio Grande do Sul, located in southern Brazil, wetlands are being converted to agricultural uses such as rice cultivation.Approximately 73% of Brazil's 1.3 million hectares under rice cultivation are in Rio Grande do Sul (Primel et al. 2005).Rice cultivation contributes to surface and groundwater contamination, because agrochemicals are applied in large quantities to control weeds and to supplement mineral nutrients (FEPAM 2012).Copper accumulation in plants and the consequent phytotoxicity can be attributed to the copper content of the herbicides, fertilizers and fungicides oft en used in agriculture (Baker 1990, Mal et al. 2002, Hu et al. 2010).Because plants are known to have diff erent degrees of resistance to heavy metals and to vary in their capacity to accumulate heavy metals (Baker et al. 2000), toxic residues of agrochemicals can aff ect non-target species that naturally occur in wetlands, thereby compromising their conservation status (Terra et al. 2008;Droste et al. 2010).
Regnellidium diphyllum Lindm.(Marsileaceae) is a heterosporous fern of the Marsileaceae family.Its distribution is restricted to Southern Brazil and some adjoining areas in Uruguay and Argentina (Schultz 1949;Alonso-Paz & Bassagoda 2002).It grows in wetlands, its roots are fi xed in humid soil or mud, and its leaves are frequently subjected to fl ooding (Schultz 1949).Th e species is currently listed as "vulnerable" on the endangered species list of the state of Rio Grande do Sul (Rio Grande do Sul 2003).Considering the conservation status of this species as well as the continuous destruction and contamination of habitats, studies have been conducted to evaluate the eff ects of heavy metals and other pollutants on R. diphyllum (Wunder et al. 2009;Cassanego et al. 2010;Droste et al. 2010;Kieling-Rubio et al. 2010& 2012).
Th e objective of this in vitro study was to investigate megaspore germination and initial development of R. diphyllum sporophytes in the presence of copper, thereby providing information about the impact that copper, as a pollutant, has on the initial stages of the R. diphyllum lifecycle.

Material and methods
Mature sporocarps were obtained from diff erent plants from a natural population of R. diphyllum in the Gravataí River Basin (29°57'18"S; 51°1'52"W), in the municipality of Gravataí, which is in the state of Rio Grande do Sul.Voucher specimens were deposited at the Herbarium Anchieta (PACA), in the city of São Leopoldo, Brazil.
Fift een sporocarps were washed under tap water, aft er which they were disinfected with 70% ethanol solution for 30 seconds and 7% sodium hypochlorite solution for 10 minutes before being washed four times in sterile distilled water and dried on sterile fi lter paper.Th e sporocarps were then mechanically opened, and megaspores were separated from microspores under stereomicroscopy.Megaspores from diff erent sporocarps were mixed in order to obtain a random sample.Because apogamy can occur naturally in megagametophytes of R. diphyllum (Mahlberg & Baldwin 1975), only megaspores were used, the objective being to obtain uniform cultures without mixing sexually and apogamically formed sporophytes.All procedures were conducted under a laminar fl ow hood.
Meyer's solution was prepared for use as culture medium (Meyer et al. 1955) and its pH was adjusted to 5.5 before autoclaving.Copper was added to the culture medium in the form of copper sulphate (CuSO 4 ), at concentrations of 1, 5, 10, 50 and 100 mg L −1 .A CuSO 4 -free culture medium was used as a control solution.Megaspore germination and sporophyte development of R. diphyllum in a CuSO 4 -free medium has previously been described by Wunder et al. (2009).Megaspores were kept in glass vials (4.5 × 10 cm) containing 30 mL of Meyer's solution (n = 15 megaspores/vial).Th ree vials were prepared for each concentration of CuSO 4 .Th e experiment was conducted in a growth chamber at 25 ± 1°C, with a 12/12-hour light/ dark cycle, under fl uorescent lights, which provided a photon fl ux density of 100 μmol m −2 s −1 .Megaspores that exhibited at least the initial apical globular green structure with a crown of rhizoids were considered to be germinated (Wunder et al. 2009).In order to evaluate sporophytic development, two megaspores were taken at random from each of the vials aft er 14 and 28 days of cultivation, respectively, resulting in a total of six megaspores for each CuSO 4 concentration.Th e primary root, primary leaf and secondary leaf of each individual were measured.Chlorosis and necrosis of sporophytes were noted and recorded throughout the experiment.
Data were tested for normality using the Shapiro-Wilk test.Germination percentages and mean lengths of roots were compared using the Kruskal-Wallis test followed by the Student-Newman-Keuls test, at a probability of 5%.Mean lengths of primary and secondary leaves were compared using ANOVA followed by Tukey's test, at a probability of 5%.Linear regression analysis was applied to estimate the relationship between CuSO 4 concentrations and mean leaf lengths.Statistical analyses were conducted with the Statistical Product and Service Solutions, version 20.0 (SPSS Inc., Chicago, IL, USA) and BioEstat, version 5.0 (Sociedade Civil Mamirauá, Belém, Brazil).

Results
Megaspores germinated at all CuSO 4 concentrations.In the CuSO 4 -free culture medium, 73% of the spores germinated, whereas (at the opposite end of the spectrum) only 6% germinated in the presence of 100 mg L −1 of CuSO 4 (H 5,225 =12.255, p=0.0315), as shown in Fig. 1.
The majority of germinated megaspores developed into sporophytes with a primary root, a primary leaf with a linear lamina and 1-2 secondary leaves.Sporophyte roots in solutions containing 10, 50 or 100 mg L −1 of CuSO 4 were signifi cantly shorter than were those in the control medium, aft er 14 days (H 5,30 =18.1413, p=0.0028) and aft er 28 days (H 5,30 =23.6838, p<0.001), as can be seen in Fig. 2 (A and B, respectively).From 14 to 28 days, the mean length of the primary root did not increase at 10 or 50 mg L −1 of CuSO 4 and even decreased at 100 mg L −1 .
As shown in Fig. 2C, primary leaf development was unaffected by the presence of copper after 14 days of exposure (F 5,30 =2.663, p=0.051).Aft er 28 days (Fig. 2D), there was a signifi cant negative relationship between CuSO 4 concentration and primary leaf length (R 2 =0.118,F=5.367, p=0.026) although the diff erence in relation to the control was signifi cant only at 100 mg L −1 (F 5,30 =10.961, p<0.001).
Secondary leaf growth also correlated signifi cantly with CuSO 4 concentration aft er 14 days of exposure (R 2 =0.374,F=20.342, p<0.001) and aft er 28 days of exposure (R 2 =0.553,F=42.143, p<0.001).At 14 days (Fig. 2E), secondary leaves of sporophytes grown in 100 mg L −1 of CuSO 4 were signifi cantly shorter than were those of sporophytes grown in the control solution and in the solutions with lower CuSO 4 concentrations (F 5,30 =6.041, p=0.001).At 28 days (Fig. 2F), secondary leaves of sporophytes grown in ≥ 5 mg L −1 of CuSO 4 were progressively shorter than were those formed of sporophytes grown in the control solution (F 5,30 =14.344, p<0.001).Most of the plants exposed to the highest concentration of copper showed signs of toxicity, such as leaf chlorosis and root necrosis.

Discussion
Copper acts as a structural element in regulatory proteins, as well as participating in photosynthetic electron transport, mitochondrial respiration, oxidative stress responses, cell wall metabolism and hormone signaling (Marschner 1995).Nevertheless, in excess, copper can cause disorders in plant growth and development, aff ecting key enzymes and altering nitrogen metabolism (Yruela 2005;Soudek et al. 2010).
Th e low proportion of R. diphyllum megaspores observed here has also been reported in previous in vitro experiments with other metals.Wunder et al. (2009) observed that increasing cadmium levels to 50 mg L −1 in culture medium resulted in a low R. diphyllum germination rate (58%) and that no germination occurred at 100 mg L −1 .Kieling-Rubio et al. (2010) observed that 50 mg L −1 of hexavalent chromium also had a signifi cant negative impact on R. diphyllum germination.Th ose same authors later found that nickel also reduced R. diphyllum spore germination, although the germination rate at 100 mg L −1 was still 46%, higher than that observed here for the same concentration of copper (Kieling-Rubio et al. 2012).
We found that sporophyte development, especially root growth and the development of the secondary leaf, was negatively infl uenced by excess copper.Th e decrease in the mean length of the primary root observed at the highest CuSO 4 concentration and longest exposure time correlated with senescence and necrosis in root tissue, as previously reported by Kieling-Rubio et al. (2012) for R. diphyllum exposed to nickel.Roots absorb metals directly from the medium and accumulate those metals (Baker 1990), and such accumulation can therefore cause irreversible damage even in the initial stages of development (Kieling-Rubio et al. 2010).
Plants grown in the presence of high levels of copper can exhibit reduced biomass and chlorotic symptoms (Yruela 2005).Th e consequences of excess copper include reduced chlorophyll content, as well as abnormalities of chloroplast structure and thylakoid membrane composition (Baszynski et al. 1988;Quartacci et al. 2000).High levels of copper can also aff ect the uptake of other elements, such as nitrogen, phosphorus, potassium, calcium, magnesium, manganese, zinc and, in particular, iron (Foy et al. 1978;Malavolta et al. 1997).Interactions between and among elements are oft en complex and dependent on the plant species involved, the concentrations of elements and the pH of the nutrient solution (Xia & Shen 2007;Koppitke et al. 2010).
Sporophytes of R. diphyllum are also sensitive to other metals in nutrient solution.Wunder et al. (2009) demonstrated that primary root growth and primary leaf growth are signifi cantly reduced and that no secondary leaves develop when R. diphyllum is grown in solutions with cadmium concentrations ≥ 12.5 mg L −1 .Hexavalent chromium has also been shown to be toxic to R. diphyllum, stunting the growth of roots and leaves, at concentrations ≥ 3.2 mg L −1 (Kieling-Rubio et al. 2010).Kieling-Rubio et al. (2012) found that the primary roots, primary leaves and secondary leaves of R. diphyllum were all signifi cantly shorter when grown in solutions with nickel concentrations of 3.2 or 4.8 mg L −1 than when grown in a nickel-free solution, and that the leaves of plants grown in the 4.8 mg L −1 solution exhibited chlorosis and necrosis.
Th e negative impact of copper on the growth of plantlets, such as that reported here for R. diphyllum, has also been observed for other species cultivated in nutrient solutions.Copper concentrations ≥ 3 mg L −1 have been shown to cause progressive stunting of growth and reduced CO 2 assimilation in the aquatic fern Salvinia minima Baker, a species in which the accumulated concentration of copper was found to be 100 times greater than that normally present in aquatic ecosystems (Al-Hamdani & Blair 2004).Mal et al. (2002) demonstrated that Elodea canadensis L. C. Rich. in Michx.plants grown for 25 days in nutrient solutions containing 5 or 10 mg L −1 of copper exhibited shorter shoots and lower dry mass than did those grown in copper-free solutions.In the angiosperm Elsholtzia Willd., hypocotyl and radicle lengths have been shown to be signifi cantly reduced (compared with controls) in plants cultivated for 10 days in solutions containing 50 or 100 μmol L −1 of copper (Xia & Shen 2007).Michaud et al. (2008) observed severe phytotoxicity symptoms in hydroponic cultures of Triticum turgidum durum L. treated with > 1 μmol L −1 of copper.However, comparing metal toxicity across studies is oft en diffi cult due to diff erences in experimental conditions, which can aff ect the concentrations that are considered toxic.
Th e area from which the sporocarps were collected for the present study is used for agricultural activities, primarily rice cultivation (FEPAM 2012).Th erefore, it is to be expected that signifi cant quantities of herbicide, fertilizer and fungicide residues would be present in the environment, contributing to copper accumulation and phytotoxicity (Hu et al. 2010).Considering the negative impact of copper on R. diphyllum germination and sporophyte development, we can posit that contamination of aquatic environments resulting from agricultural activities, especially rice cultivation, poses a risk to the establishment and conservation of natural populations of this already vulnerable species.

Figure 1 .
Figure 1.Germination of Regnellidium diphyllum Lindm.megaspores in Meyer's solutions containing a range of copper sulphate (CuSO 4 ) concentrations aft er 28 days.* indicates a signifi cant diff erence between the treatment and the control (CuSO 4 -free solution) according to the Student-Newman-Keuls test.Error bars indicate standard deviations.

Figure 2 .
Figure 2. Growth of Regnellidium diphyllum Lindm. in Meyer's solutions containing a range of copper sulphate (CuSO 4 ) concentrations. A. Primary root length aft er 14 days.B. Primary root length aft er 28 days.C. Primary leaf length aft er 14 days.D. Primary leaf length aft er 28 days.E. Secondary leaf length aft er 14 days.F. Secondary leaf length aft er 28 days.* indicates a signifi cant diff erence between the treatment and the control (CuSO 4 -free solution) according to the Student-Newman-Keuls test (A-B) and Tukey's test (C-F).Error bars indicate standard deviations.