In vitro Evaluation of Copper Tolerance and Accumulation in Populus nigra

Phytoextraction is an efficient and cheap way to extract copper from soils in riparian zones. In this work five genotypes of the endangered tree species Populus nigra L. were tested for their copper tolerance and accumulation in vitro when cultivated on media with three Cu concentrations: 10 -3 , 10 -4 and 10 -7 M (buffered with citric acid/Na-citrate buffer, pH 3 before sterilization). After five-weeks cultivation of rooted shoots, the highest increases in morphological and biomass parameters were observed at 10 -7 M Cu 2+ . As the medium with 10 -3 M Cu 2+ exhibited a toxic effect, the effect of 10 -4 M Cu 2+ and pH 3 was used for further genotype evaluation. According to the measured morphological and parameters of photosynthetic pigment contents, the best performance was achieved by the genotype Populus nigra cl. DN3. The highest copper accumulation on the same medium was achieved by genotype Populus nigra cl. BN5. The obtained data point to the considerable potential of the applied method in the evaluation of Populus nigra genotypes for use in projects of copper phytoextraction. https://doi.org/10.2298/ABS170210014V Received: February 10, 2017; Revised: April 6, 2017; Accepted: April 26, 2017; Published online: May 15, 2017 How to cite this article: Vuksanovic V, Kovacevic B, Katanic M, Orlovic S, Miladinovic D. In vitro evaluation of copper tolerance and accumulation in Populus nigra . Arch Biol Sci. 2017;69(4):679-87.


INTRODUCTION
Heavy metals are among the most important pollutants threatening the ecosystem.Human habitable zones contain heavy metals that are potentially harmful to human health, as well as that of plants and other living organisms [1].The toxic effect of most heavy metals is caused by their bonding to protein sulfhydryl groups, which leads to the inhibition of enzyme activity, compromises protein structure and causes the substitution of essential elements in biomolecules [2]; high concentrations of metal ions in the soil limit the assimilation of important micro-and macronutrients by plants [3,4].
Copper is an essential microelement, necessary for metabolism in all living organisms.However, excess concentrations of copper in plants can cause problems in the formation of the root system and plant growth in general.This effect is increased in soils with a fine texture and high cation exchange capacity because they contain more organic matter and have a low pH [5].Recent research has described the accumulation of copper in alluvial zones [6], zones around mine sites, areas polluted after accidents in mine facilities [7] and agricultural soils treated with sewage sledge [8].Thus, phytoremediation projects are of interest as they could improve cultivation in copper polluted areas by stabilizing this pollutant in the soil, thus preventing its excess in groundwater and its spread by wind; they could also be used to extract copper from the soil and thereby lower its presence.Research on the influence of different plant species on contaminated soils and underground water began in the early 1980s [9][10][11].Trees have been suggested as a low-cost, sustainable and ecologically sound solution for the remediation of heavy metal-contaminated land [12], especially by phytoextraction [13].
The reaction of plants to excessive concentrations of copper was commonly tested on herbaceous species; however, recently there is an interest in the use of tree species (mainly hybrid poplars) in the management of copper contaminated areas [14].Poplars are tree species that are often used in phytoremediation due to their rapid growth, adaptability, well-developed root system that reaches underground waters, and the ability to transpire considerable amounts of water [15].Poplars are not hyperaccumulators, but because of their large biomass production [12] and relatively high quantity of extracted metal per plant [13], potentially large quantities of heavy metals could be extracted.The European black poplar is of special interest as an endangered indigenous species that could be used in riparian areas under different protection regimes.The use of this species in phytoremediation projects in riparian zones could also contribute to improving the stability and biodiversity of this species and related ecosystems [16].
Aside from its importance as a rapid means of producing clonal planting stock, in vitro culture of tree species can also facilitate studies on the effects of elevated heavy metal concentrations and on the selection of tolerant genotypes.The molecular data obtained by Castiglione et al. [17] in the white poplar suggest that the in vitro model is a sensitive and reliable system for the study of responses to heavy metal stress.This is an important fact when considering the difficulties encountered in experiments on large longlived organisms.Heavy metal tolerance and accumulation of the European black poplar has been poorly tested in vitro and needs further research.
In this work, five different European black poplar (Populus nigra L.) genotypes were studied in vitro for their tolerance to copper based on morphological parameters, biomass accumulation and pigment content, as well as copper accumulation in aboveground plant parts.Also, the effect of the pH of the medium and copper concentration were examined in order to optimize the evaluation of copper tolerance in vitro.The aim of our work was to examine and select copper tolerant and accumulating genotypes, which are potentially interesting for copper phytoextraction in soils contaminated with copper.

Copper treatments
For the experiment, 1.5-2.0-cm-longshoot tips of the previously multiplied shoots were placed on a rooting medium based on ACM supplemented with 0.1 μM 2,3,5-triiodbensoic acid (TIBA), 0.1 μM IBA, 26.64 μM glycine and 1.2 mM citric acid, 0.9% agar and 58.43 mM sucrose.The effects of the following three Cu 2+ concentrations were examined: 10 -7 , 10 -4 and 10 -3 M, in media labeled C1, C2 and C3, respectively, pH 3 (adjusted before sterilization).The control medium (C0) contained 10 -7 M Cu 2+ , pH 5.5.Sterilization was performed using a microwave oven.The media were treated until they started to boil, and were then poured into sterilized jars in a laminar chamber.In this way, the jellification potential of agar was preserved in media with pH 3 [21].A citric acid/Na-citrate buffer was used to provide pH stability [22].Citric acid is expected to improve copper import into plants and in this way it provides a more critical test for copper tolerance and accumulation.Also, this buffer system provided a relatively wide pH spectrum for testing.The cultures were maintained in the same conditions as previously described for multiplication.Also, before placing into the experimental media, the plants were cultivated for two weeks on the control medium (C0) in order to eliminate the influence of cytokinins from the multiplication medium.For the experiment, three jars with five plants per jar were set per each combination of genotype×medium in three repetitions.For pigment content determination, an additional three jars with five shoot per jar were established per each combination of genotype×medium.

Copper tolerance assessment
After 35 days of culture, the following characters were determined: morphological characters: number of roots per shoot and shoot height; characters describing the biomass and water content: fresh shoot mass per plant, dry shoot mass per plant, the shoot moisture content; characters describing the contents of photosynthetic pigments in fresh shoot mass: the contents of chlorophyll a (Chl a), chlorophyll b (Chl b), total carotenoids, chlorophyll a and b (Chl a+b), the chlorophyll a/b ratio.The concentrations of chloroplast pigments (Chl a, Chl b and total carotenoids) were determined spectrophotometrically [23].The Chl a+b and chlorophyll a/b ratios were calculated.

Copper accumulation assessment
Two characters of copper accumulation were determined: copper accumulation (copper content per shoot dry mass) and copper content (copper content per shoot).In order to determine copper accumulation, i.e. the copper concentration in dry biomass (mg kg -1 ), samples were mineralized by wet-washing in a microwave digester in 65% HNO 3 and 30% H 2 O 2 (5:1 v/v).The copper content in a sample was determined by atomic absorption spectrometry (AA 240FS Fast Sequential Atomic Absorption Spectrometer, Varian, Australia).

Statistical analysis
The entire experiment was designed to be completely randomized.The data for the number of roots were transformed by square transformation ( 1 + X ), and the data for the percentage of rooted shoots by arcsine transformation (

X arcsin
).These transformations were conducted in order to meet the normal distribution of frequencies of data required for the implemen-tation of the used statistical methods.The obtained data were analyzed by two-way ANOVA and the LSD test with R statistical program [24].

RESULTS
According to the results of ANOVA, the differences among genotypes were significant only in the morphological and biomass characters, with the exception of dry root mass.However, the examined media had a significant effect on the variation of all examined characters, except dry shoot mass, whereas the interaction genotype×medium had a significant influence on copper accumulation characters and some characters of the photosynthetic pigment content (Table 1).

Morphological characters
There is a considerable difference in the effects of the media on the examined genotypes.Chlorosis, necrosis and absolute absence of root formation were   The differences among values marked with the same letter are not significant at the level α=0.05 observed on C3 medium in all genotypes.The best rooting performance was achieved on C1 medium, while there was no significant effect of media on shoot height (Table 2).
The best performance according to all four examined morphological characters was achieved by genotype DN3, while the poorest results for the morphological characters was achieved by PN2.Although the effect of the interaction genotype x medium was not significant, GN5 showed a specific response on C1 medium that was not significantly different from that obtained on the control (C0) (Table 2).
Other tested genotypes achieved significantly higher morphological characters on C1 than on C0.For medium C2, the results were considerably lower than on the control, while on medium C3 growth was almost absent.Only genotype DN3 attained significant results with regard to morphological characters on C2, which was at the level of the control.

Biomass characters
Although genotype DN3 had the highest values for dry root and shoot mass, differences among the examined genotypes in biomass characters were not statistically significant.However, there were differences in the reaction of the genotypes on the examined media.DN3 achieved a significantly higher shoot dry mass on C2 than on C1, while GN5 achieved higher dry root mass on C1 than on other media (Table 3).
In general, there were no significant differences between the treatments and the control for most of the examined biomass characters.Shoot moisture content and root/shoot dry mass ratio were significantly lower on the medium with the toxic copper concentration (C3).Furthermore, significantly greater root dry mass and root/shoot dry mass ratio were observed on the C1 medium than on C2 (Table 3).

Photosynthetic pigment content
The genotype DN3 had significantly higher photosynthetic pigment contents than the other genotypes.It also differed from the others by its reaction on the media.For this genotype, the pigment contents on C2 were significantly higher than on the control, while the differences were not significant in the other genotypes (Table 4).The photosynthetic pigment content calculated for fresh mass revealed almost the same relations among the genotypes and media (data not shown).
The highest content of photosynthetic pigments was achieved on C2, and the lowest on C3.However, only the content of Chl b on C2 significantly differed The differences among values marked with the same letter are not significant at the level α=0.05 Table 3. continued from C0. Also, only on C3 were the carotenoid content and chlorophyll a/b ratio significantly lower than on the other media.

Copper accumulation and content
There were no significant differences in copper accumulation and content between C0 and C1, while C2 and C3 differed significantly from C0 and C1 in both characters (Table 5).The highest value of copper accumulation and copper content in most genotypes was observed on C3 medium, except for BN5, which displayed the highest copper accumulation and content on C2.

DISCUSSION
High copper tolerance and the ability to accumulate this metal in the aboveground parts of plants are principal criteria in the evaluation of genotypes in order to be considered for use in phytoextraction [13].Di Lonardo et al. [13] established that woody plant medium (WPM) with 10 -3 M Cu 2+ and pH 5.2 had no inhibitory effect on the growth and development of white poplar genotypes.In our work we conducted the test at pH 3, using citric acid to lower and stabilize the pH.It is well known that the pH of the media could be altered by many factors during both sterilization and cultivation in vitro [21].Citric acid  Lables of examined media: C0 -10 -7 Cu 2+ , pH 5.5, C1 -10 -7 Cu 2+ , pH 3, C2 -10 -4 Cu 2+ , pH 3, C3 -10 -3 Cu 2+ , pH 3 before autoclaving 2 The differences among values marked with the same letter are not significant at the level α=0.05 is also known as a low molecular weight organic acid (LMWOA) capable of forming chelates with heavy metals, improving their mobility and bioavailability without causing leaching of heavy metals into lower soil strata [25,26].According to Evangelou et al. [26], the effect on copper bioavailability in research on Nicotiana tabacum was even better with citric acid than with EDTA.Chen et al. [27] showed that the effect of citric acid on the uptake of lead and cadmium, anions that have even less mobility, is strongly related to pH lowering.In our work, in presence of citric acid (1.2 mM) and low pH, the concentration of 10 -3 M Cu 2+ in medium C3 was sufficient to produce a toxic effect in the examined European poplar genotypes.Therefore, a copper concentration of 10 -4 M in media with pH 3 should be recommended in future work on copper tolerance and the evaluation of its accumulation in Populus nigra in vitro.In addition, these results should be taken into consideration in further research in field conditions on acidic soils and in cases when citric acid is used to improve copper availability.
The effect of low pH was tested by comparing the results of C1 and C0.The number of roots and the percentage of rooted shoots were significantly higher on C1; however, the shoot height and shoot dry mass on this medium were similar to those on C0.Kovačević et al. [21] observed significant growth and improved development of white poplar with regard to both shoot and root in plants grown in vitro on medium with an initially low (before sterilization) pH of 3.0.The authors did not use citric acid nor any additional buffer system.At the end of cultivation, the final pH of the media differed significantly from the initial value.The Na-citrate/citrate buffer system together with microwave sterilization could be a useful approach for further studies of the effect of low pH in vitro.
Among the examined morphological characters, the best differentiation among genotypes on C2 medium was observed on root formation, in particular the rooting percentage.Genotype DN3 had the highest scores for rooting characters, which points to a high tolerance to increased copper concentration of this genotype.Greater differences in rooting characters than in shoot height between the among genotypes could be related to the fact that the roots of poplar genotypes in vitro seem to be more sensitive to high heavy metal concentrations in media compared to the shoots [13].It seems that morphological differences in the response to C2 medium among the genotypes were most intensively expressed by their most sensitive organ, the root.Thus, we assumed that the rooting characters could be proposed for quick copper tolerance in vitro tests in Populus nigra in the future.There were significant differences among genotypes in photosynthetic pigment contents, especially on the C2 medium.On this medium, genotype DN3 differed significantly from the others, with higher chlo- The differences among values marked with the same letter are not significant at the level α=0.05 rophyll and carotenoid contents, while the genotype PN2 had the highest chlorophyll a/b ratio (Table 4).
In our study, chlorosis was observed on C3 medium in all genotypes.The loss of photosynthetic pigments is a common reaction of plants to excessive copper, and is related to disturbances in the chloroplast inner structure caused by alterations in lipoproteins in thylakoid membranes [29].No significant difference in the content of examined photosynthetic pigments were observed on C2 compared to C0 and C1, whereas the chlorophyll a/b ratio was significantly lower in samples grown on the C3 medium than on C1.In contrast to our results, Borghi et al. [14] observed an increment in the chlorophyll a content in treatments with copper concentrations ranging from 0.4 to 500 10 -6 M Cu 2+ , which was followed by a significant decline in Populus × euramericana cl.Adda leaves after growth in 10 -3 M Cu 2+ in hydroponic culture.The same author found that the excess of copper produced a significant difference in the chlorophyll a/b ratio of the rooted cuttings.
In general, copper accumulation increased with the increment of copper concentration in the medium.In the Euramerican poplar clone Adda grown in hydroponics, Borghi et al. [14] found such an increment only in roots, but not in the stem and leaves.The highest copper accumulation and copper content in shoot tissue of the examined European black poplar genotypes was observed on C2 and C3 media.
We propose the C2 medium for application in further tests of copper tolerance and accumulation in Populus nigra tissue culture, since the C3 medium had a toxic effect on all tested genotypes.The C2 medium had an inhibitory but not toxic effect, and significant differences in most of the examined characters as compared to the control medium.Furthermore, it provided the best differentiation among the genotypes.
The examined genotypes considerably differed in copper tolerance and accumulation.Genotype DN3 achieved the best performance according to morphometrics, biomass and photosynthetic pigment contents.High scores in morphometrics, especially in rooting characters and in the photosynthetic pigment contents, on C2 suggest that this genotype could tolerate the presence of copper in the substrate at increased concentrations.The highest copper accumula-tion on C2, the medium preferred for copper tolerance evaluation tests, was achieved by BN5, while all the other genotypes had the highest copper accumulation when grown on the C3 medium.These results favor BN5 to be tested beside DN3 in phytoextraction projects on soils where the copper content is low enough to be sufficiently tolerated by this genotype.Here it could achieve a higher copper accumulation than the other genotypes and would be more appropriate for copper phytoextraction on soils with a near to toxic copper concentration.Further field tests should be performed in order to examine the effect of copper on biomass accumulation, which is the other component of a plant's phytoextraction potential.The use of Populus nigra genotypes on highly contaminated soils should be done with caution, especially considering the inhibitory effect of high copper concentration on root formation.
The general opinion is that differences in the bioavailability of contaminants and the processes of pollutant uptake and metabolite distribution are likely to be substantial in tissue culture and field conditions.In Salix sp. it was shown that the results obtained in hydroponics and in the field are comparable [29,30].Also, in [31] and [32], the authors support the idea that the response of plants to environmental contaminants can be predicted based on results from tissue cultures, reducing the cost of subsequent conventional whole-plant experiments.
Considering the observed differences among the examined genotypes with regard to copper tolerance and accumulation, in vitro tests can serve to narrow the group of candidate genotypes for copper phytoextraction projects.However, for the final evaluation of a particular genotype, research should be performed in the field, considering the lower availability of lead in soil, higher juvenility of the material in vitro and complexity of the interaction between plant and the habitat.

Table 1 .
F-test of ANOVA for the examined characters.

Table 2 .
Morphological characters of rooted shoots of Populus nigra grown on the examined media (LSD test). ef16.74

Table 3 .
Biomass characters of rooted shoots of Populus nigra grown on examined media (LSD test).

Table 4 .
Photosynthetic pigments' content in dry shoot mass of rooted shoots of Populus nigra grown on examined media (LSD test).

Table 5 .
Copper accumulation and content in rooted shoots of Populus nigra grown on examined media (LSD test).