Ecotoxicology and Environmental Safety 148 (2018) 675–683

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Ecotoxicology and Environmental Safety

journal homepage: www.elsevier.com/locate/ecoenv

Genetic characterization, micropropagation, and potential use for arsenic T

phytoremediation of Dittrichia viscosa (L.) Greuter
Francesco Guarinoa,1, Barbara Conteb,1, Giovanni Improtac, Rosaria Sciarrillob,
⁎
Stefano Castiglionea, Angela Cicatellia, , Carmine Guarinob
a
Department of Chemistry and Biology “A. Zambelli” University of Salerno, Via Giovanni Paolo II, 132, 84084 Fisciano, Salerno, Italy
b
Department of Science and Technology, University of Sannio, Via Port’Arsa, 11, 82100 Benevento, Italy
c
Department of Public Health, University of Napoli Federico II, Via Pansini 5, 80131 Napoli, Italy

A R T I C L E I N F O A B S T R A C T

Keywords: In the last decade, many scientists have focused their attention on the search for new plant species that can offer
Dittrichia viscosa improved capacities to reclaim polluted soils and waters via phytoremediation. In this study, seed batches from
Genetic study three natural populations of Dittrichia viscosa, harvested in rural, urban, and industrial areas of central and
In vitro culture southern Italy, were used to: (i) evaluate the genetic and morphological diversity of the populations; (ii) develop
Arsenic pollution
an efficient protocol for in-vitro propagation from seedling microcuttings; (iii) achieve optimal acclimatization of
Phytotechnology
micropropagated plants to greenhouse conditions; (iv) test the response to arsenic (As) soil contamination of
micropropagated plants. The genetic biodiversity study, based on Random Amplification of Polymorphic DNA
(RAPD), as well as the morphometric analysis of 20 seedlings from each population revealed some degree of
differentiation among populations. Based on these data, the most biodiverse plants from the three populations
(10 lines each) were clonally multiplied by micropropagation using microcuttings of in-vitro grown seedlings.
Three culture media were tested and Mureshige and Skoog medium was chosen for both seedling growth and
micropropagation. The micropropagated plants responded well to greenhouse conditions and over 95% survived
the acclimatization phase. Four clones were tested for their capacity to grow on soil spiked with NaAsO2 and to
absorb and accumulate the metalloid. All clones tolerated up to 1.0 mg As. At the end of the trial (five weeks), As
was detectable only in leaves of As-treated plants and concentration varied significantly among clones. The
amount of As present in plants (leaves) corresponded to ca. 0.10–1.7% of the amount supplied. However, As was
no longer detectable in soil suggesting that the metalloid was taken up, translocated and probably phytovola-
tilized.

1. Introduction (e.g., phyto-stabilization, -volatilization, -extraction), have become of

In the last two centuries, industrialization has led to a large increase
in heavy metals (HMs; Cd, Pb, Zn etc.) and metalloids (Ms; As, Se, etc.)
in different environmental matrices representing a serious concern for
the environment and for human health (Adriano, 2001; Miguel and
Marum, 2011; Perez-Sirvent et al., 2012; Fernandez et al., 2013). Al-
though some HMs and Ms are essential for plant growth, at high con-
centrations they are toxic, causing deleterious effects on several meta-
bolic processes (Emamverdian et al., 2015; Pistelli et al., 2017). In fact,
even Ms, such as arsenic (As), are considered extremely toxic for all
living organisms (Abioye, 2011). The application of green technologies,
such as phytoremediation, and of all the technologies associated to it
common use and are well accepted by the public opinion, politicians
and decision makers (Singh et al., 2015).
Phytoremediation, in general, refers to the use of plants for the re-
moval of HMs and Ms from soil or water. In particular, phytostabil-
ization is based on the ability of plants, and their root-associated mi-
croorganisms, to immobilize HMs and Ms in the roots and/or in the
rhizosphere. Phytoextraction involves the translocation of con-
taminants from soil and roots to the above-ground organs of the plant;
phytovolatilization is the ability of a plant to take up pollutants,
translocate, chemically modify, and, finally, volatilize them via the
transpiration stream. In the past two decades, plant biologists and
ecologists alike have been searching for species with high

⁎
Corresponding author.
E-mail addresses: fguarino@unisa.it (F. Guarino), bbconte@yahoo.it (B. Conte), ing.improta@gmail.com (G. Improta), sciarrillo@unisannio.it (R. Sciarrillo),
scastiglione@unisa.it (S. Castiglione), acicatelli@unisa.it (A. Cicatelli), guarino@unisannio.it (C. Guarino).
1
Equal contribution.

https://doi.org/10.1016/j.ecoenv.2017.11.010
Received 31 May 2017; Received in revised form 3 November 2017; Accepted 5 November 2017
Available online 21 November 2017
0147-6513/ © 2017 Elsevier Inc. All rights reserved.
F. Guarino et al.
phytoremediation capacity to use them for the reclamation of HM- and
Ms-polluted soils ( Nogales and Benítez, 2006; Jimenez et al., 2011).
Only a few studies have described Dittrichia viscosa (L.) Greuter as a
good candidate for phytoremediation purposes. In Italy, this species
was found growing spontaneously at the former “Pertusola Sud” Zn-
smelter plant (Marchiol et al., 2013) and on an ex-mining site on the
island of Elba (Pistelli et al., 2017). It is a ruderal shrub of the Aster-
aceae family (Inuleae tribe, also known as Inula viscosa L.) and is con-
sidered an invasive plant in all the northwestern Mediterranean basin
due to its genetic plasticity and adaptability to different soils (ranging
from siliceous to calcareous) and climatic conditions (Araniti et al.,
2017). It is also considered a ruderal and pioneer species for its ability
to thrive in disturbed areas (e.g., mining, industrial, civil, agriculture,
etc.) and under stressful environmental conditions, such as large
changes in humidity and salinity (Curadi et al., 2005; Batista et al.,
2013). Conesa et al. (2011) indicated D. viscosa as a species able to
accumulate large quantities of HMs and Ms from sand dunes polluted by
mining wastes. Moreover, Barbafieri et al. (2011) reported D. viscosa as
one of the species with the highest concentration of trace elements in its
aboveground biomass among plants growing on ex-mining soils in
Sardinia, suggesting that this species can be considered a good “phy-
toextractor”.
Several studies have suggested that for conducting efficient soil
reclamation it is better to use stable and selected genotypes and clones;
to this purpose, in-vitro micropropagation can be considered a good tool
to obtain clonal plants. To our knowledge, selection of D. viscosa gen-
otypes useful for phytoremediation purposes and their clonal propa-
gation has not been reported.
The aims of our work were to: (i) evaluate the genetic and mor-
phological diversity of three different seed batches collected from nat-
ural populations of D. viscosa, growing spontaneously in several Italian
locations (rural, urban, and industrial areas); (ii) develop an efficient
seed surface sterilization and micropropagation protocol; (iii) evaluate
the response of micropropagated clones in soils spiked with As.
2. Materials and methods
2.1. Plant material
Mature inflorescences of D. viscosa were harvested from adult plants
of three natural populations (named A, B, and C) growing in several
Italian locations all characterized by a typical Mediterranean climate.
Population A is located along the roadside in a ruderal area (Laurentina,
Rome-IT) whose soils derive from alteration of volcanic rocks, popu-
lation B grows on sandstone soils (Corleto Perticara, Potenza-IT), and
population C grows on a multi-contaminated site in which both in-
organic and organic pollutants are present [Crotone, IT; (Marchiol
et al., 2013)].
2.2. Genetic characterization of D. viscosa specimens
Genomic DNA was extracted from 200 mg fresh weight leaves of
each plant (20 individuals per population) using the CTAB method
(Doyle, 1991). The nucleic acid quality, integrity, and quantity were
Table 1
Genetic variation, based on RAPD analysis, within groups of D. viscosa individuals from populations A, B, and C.
Specimen group N Ne
A 1.83 1.58
B 1.70 1.56
C 1.96 1.69
Mean ± Standard Errors 1.83 ± 0.045 1.61 ± 0.038
N = mean number of alleles per locus, Ne = effective number of alleles per locus, P = percentage of polymorphic loci (95% criterion), He = Nei (1973) unbiased expected gene
diversity, I = Shannon's index over loci. Standard errors are given in parenthesis.
676
Ecotoxicology and Environmental Safety 148 (2018) 675–683
estimated on agarose gels and spectrophotometrically (NanoDrop,
Thermo Scientific, US). RAPD markers were produced in a reaction
mixture of 20 μl containing: 5.0 μl (100 ng) of genomic DNA, 0.4 μl of
diluted OPA primer (10 pmols μl−1; for primer sequences see
Supplementary materials, Table SM1), 2.0 μl 10X Taq Buffer,2.0 μl
dNTP mix (2 mM for each deoxynucleotide), 1.6 μl MgCl2 (25 mM),
0.2 μl red Taq DNA Polymerase (5 U μl−1), and 8.8 μl nuclease-free
water.
The PCR reaction was performed using the following thermal pro-
file: 3 min at 94 °C; 40 cycles of denaturation at 94 °C for 1 min, an-
nealing at 40 °C for 1 min, extension at 72 °C for 2 min. PCR products
were electrophoresed on 1.5% agarose gels stained with ethidium
bromide at 100–120 v/cm for 60 min and visualized under a UV trans-
illuminator.
A binary matrix was constructed attributing a value of 1 to the
presence of each allele of a certain size at each analyzed RAPD locus,
and 0 to its absence. A similarity matrix based on the Jaccard index was
elaborated in order to draw a dendrogram using the unweighted Pair
Group Method (UPGMA; using the SAHN program in NTSYS-9pc pro-
gram ver.2.1 Exeter Software Setauket, NY – USA).
The GENALEX 6.5 software package was used to assess the genetic
diversity within groups of D. viscosa genotypes deriving from seeds of
populations A, B, and C as the proportion of polymorphic loci (P, using
the 95% criterion), mean number (N) and effective number (Ne) of
alleles per locus, Nei's gene diversity index [(Nei, 1973) - the He index
was adopted assuming the populations to be in Hardy-Weinberg equi-
librium, although we were not able to investigate this since dominant
markers were used], and Shannon's information index. The distribution
of genetic diversity within and among groups was assessed using Nei's
genetic differentiation degree [GST and Fst; (Marks, 1988)]. The Ana-
lysis of Molecular Variance (AMOVA), based on squared Euclidean
distances between all pairs of RAPD phenotypes, was calculated using
the Arlequin software (Excoffier and Lischer, 2010). The AMOVA pro-
cedure was performed in order to further partition the total genetic
variation among and within groups of genotypes.
2.3. Seed sterilization
Three different seed sterilization methods (Table 1) were tested
(five times) on D. viscosa seeds (about 200). Sterilized seeds were sown
on three different agar-soldified media: Murashige and Skoog (MS;
4.4 g of MS basal medium containing vitamins, 30 g L−1 sucrose and
8 g L−1 agar - all Duchefa Biochemie, Haarlem, The Netherlands);
Gamborg (3.2 g of Gamborg basal medium, 30 g L−1 sucrose and
8 g L−1 agar; pH 5.7 with KOH); Mohr [1 mL of Bold Basal Medium
(Nichols, 1973), 100 mg L−1KNOз, 10 mg L−1 CaCl2*H2O, 10 mg L−1
MgSO4,136mgL−1 K H2PO4, 0.4 mg L−1 FeSO4,8 g L−1 agar; pH 7.5
(Basile et al., 2013)]. All media were sterilized at 103.5 kPa at 121 °C
for 20 min.
2.4. Micropropagation from in-vitro grown seedlings
Ten sterilized seeds of each population were placed on 80 mL of MS,
Gamborg or Mohr agarized culture medium previously distributed in
P He I
83.3 0.32 0.46
76.7 0.32 0.46
96.7 0.39 0.57
85.6 ± 5.9 0.34 ± 0.026 0.49 ± 0.019
F. Guarino et al.
sterile culture boxes (Sigma-Aldrich, Milano-Italy). Seeds were in-
cubated in a growth chamber under the following conditions: 16 h
light/8 h dark with a light intensity of 50 μmol m−2 s−1 at
25 °C ± 1 °C.
Seedlings (8–10 cm high) were used to produce stem microcuttings
(about 1.5-cm long), each having an apical or nodal bud. Microcuttings
were transferred to fresh, hormone-free and agarized MS, Gamborg or
Mohr culture medium, where they rooted (for ca. two months).
2.5. Acclimatization and leaf morphometric analysis of micropropagated
plantlets
After two months of in-vitro culture, plantlets were about 10 cm high
and well rooted. These plants were recovered from the culture boxes,
their basal portions washed thoroughly under running tap water to
remove culture media residues, and then rinsed with distilled water.
The plantlets were then transferred to pots (0.25 L) filled with peaty
garden soil; they were covered with plastic bags for ca. ten days to
protect them from dehydration and obtain a gradual acclimation to
greenhouse conditions. Plants were maintained in a greenhouse with a
photoperiod of 16 h light/8 h dark (Alfonso et al., 2017).
Three fully developed basal leaves were harvested from 30 ap-
proximately six-months old micropropagated plants. Length, width,
angle of the ribs, and the presence of serrated edges were recorded for
each individual. Based on the length (L)/width (l) ratio of the leaf
blade, leaf shape was defined as follows: elliptical, if L/l < 4 cm; el-
liptic-lanceolate, if L/l = 4–6 cm; lanceolate, if L/l > 6 cm. The length
of the leaf blade was defined as follows: short, when L < 5 cm; medium,
when L = 5.7 cm; long, when L > 7 cm. The width of the leaf blade was
defined as follows: narrow, when L < 1 cm; medium, when L =
1–1.5 cm; wide, when L > 1.5 cm.
2.6. Greenhouse experiment with As
On the basis of genetic biodiversity, four micropropagated lines
(A17, B6, C3, C17) were cloned. Three plants per line were transferred
to plastic pots containing ca. 2 kg dry weight garden soil (one plant per
pot). The soil element content is shown in Table SM2. The concentra-
tions of all detected elements were lower than the legal threshold values
established by the Italian Ministry of the Environment for soils of
public, private, and residential areas (Dlgs.152/2006).
When the plants reached a height of 15 cm, they were exposed to
three treatments: unpolluted soil (CNT) and soil spiked with 0.5 mg or
1.0 mg As. In addition, 0.5 mg or 1.0 mg As spiked soils were estab-
lished without plant in order to evaluate the As volatilization due to the
greenhouse conditions. Arsenic was supplied as sodium arsenite
(NaAsO2; Sigma-Aldrich, Milano-Italy) solution (20 mg L−1) and added
to pots in consecutive weekly doses, up to the final amount of 0.5
or1.0 mg per pot. Each treatment included three pots per genotype.
2.7. Element concentration in soil and plant organs
Five weeks after soil As contamination, D. viscosa plants were har-
vested and separated into roots, stems, and leaves. The roots were
carefully washed with distilled water to eliminate soil sediments. Plant
organs from each pot and soils (from three pots) were pooled and air-
dried (room temperature) up to constant weight. Plant material was
pulverized with N2 in a mortar and, for each plant organ, three biolo-
gical replicates were analyzed.
For As determination, all materials (soil and plant organs) were
acid-digested [65% HNO3/40% HF, 2:1 (v/v)] in a microwave oven
(Ethos, Milestone) using the following digestion program:1 min at
250 W, 1 min at 0 W, 5 min at 250 W, 4 min at 400 W, 4 min at 600 W,
5 min at 250 W. Arsenic was determined by hydride generation atomic
absorption spectrometry using an atomic absorption spectrometer
(Perkin-Elmer 4110 ZL) equipped with an electrode-less discharge lamp
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Ecotoxicology and Environmental Safety 148 (2018) 675–683
(As EDL); a flow injection analysis system was used. The mineralized
samples were mixed with sodium boro-hydride (NaBH4) in sodium
hydroxide (NaOH) in order to reduce the analyte (As) to its hydride
form. For metal detection (for soil characterization and limits of de-
tection see Table SM2), soil was digested with an acid mixture [65%
HNO3: 50% HF, 2:1 (v/v)] in a microwave oven. Standard reference
material (1575a Pine Needles; NIST, 2004; Mackey et al., 2004) was
also analyzed in order to verify the accuracy of the results. Standard
solutions of each metal were used to generate calibration curves to
convert emission readings to analyte concentrations.
2.8. Statistical analysis
A Canonical Variate Analysis was performed on morphometric data
using R.2.10.1 software with a candisc package, and Pillai's elaboration
was performed on data. The data relative to element content and bio-
mass were processed by statistical tests using the SigmaPlot 12.0 soft-
ware package (Systat Software, Inc). Significant differences in biomass
production, metal soil content, and element accumulation in different
plant organs, genotypes, and treatments were tested by Kruskal and
Wallis one-way analysis of variance by ranks, followed by post hoc
Nemenyi test.
3. Results and discussion
3.1. RAPD analysis
A large variety of molecular markers, based on diverse techniques
and related to specific biological and genetic targets, has been devel-
oped. Although the utility of certain molecular markers to estimate
genetic biodiversity among and within populations and species may be
most useful than others, depending upon their purpose, in general the
choice has to be made mainly in relation to the genetic information
available for the studied species (Poczai et al., 2013). Random Ampli-
fication of Polymorphic DNA (RAPD) analysis does not require a priori
knowledge of DNA sequences and, therefore, is the method of choice to
study D. viscosa because practically nothing is known at the molecular
level for this plant species, and its genome has not yet been fully se-
quenced.
To perform the molecular analysis on all individuals of D. viscosa
populations, a total of 20 RAPD primers were tested on a limited
number of individuals; based on RAPD reproducibility, only 10 of them
(50%) produced clear, scorable and reproducible DNA bands. Five
primers (OPA 4, 5, 9, 12, 16) resulted highly polymorphic. In particular,
OPA 4, OPA 9, and OPA 12 furnished the highest number (7) of am-
plified DNA bands, while OPA 5 and OPA 16 produced the lowest
numbers (5 and 4 bands, respectively). Using these five different pri-
mers, a total of 872 RAPD bands, ranging in size from 300 to 3000 bps,
were obtained. The number of amplified fragments varied from 92
(OPA 16) to 238 (OPA 9), with 174.4 bands per primer on average (of
which 43 were polymorphic). The percentage polymorphism in D. vis-
cosa specimens varied from 85.4% to 100% (on average 89.6%). The
highest percentage was recorded for OPA 12. The number of poly-
morphic loci per primer and the percentage polymorphism detected
with the OPA primer series, used in the present study, were comparable
to those previously reported for several other plant species (Shabir
et al., 2014).
The RAPD profiles were used to calculate a similarity matrix based
on Jaccard's similarity coefficient; the software elaboration of the data
matrix produced a dendrogram of genetic similarity (Fig. 1).
The dendrogram showed two well separated groups (Groups I & II).
Group I included some individuals from population C, while Group II
comprised almost all the other analyzed plants. Group II has two sub-
clusters (IIa and IIb): cluster IIa included the remaining plants from
population C, while cluster IIb grouped all plants obtained from seeds of
population A and B. The genetic similarity ranged from 0.40 to 0.90.
F. Guarino et al. Ecotoxicology and Environmental Safety 148 (2018) 675–683

Fig. 1. UPGMA dendrogram based on RAPD profiles of D. viscosa genotypes obtained from seeds of populations A, B, and C. The dendrogram was elaborated on the basis of Jaccard's
similarity coefficient.

Ever since its development several decades ago, RAPD analysis 3.2. Seed sterilization methods and germination tests
(Williams et al., 1990) has been used for many different purposes in
evolutionary biology. Although its application in population genetic Seed contamination due to microorganisms, including viruses,
studies has been hampered by technical limitations (DNA quality, re- bacteria, fungi, is considered the most important cause of losses during
producibility, etc.), the method has been frequently adopted for fin- in-vitro axenic culture of plants. In fact, microorganisms can compete
gerprinting plant genetic resources ( Palacios and Gonzalez-Candelas, adversely with plant for nutrients of media cultures, increase plant
1997; Gonzalez-Fernandez et al., 2011; Lu et al., 2016) and for iden- mortality and also induce unstable growth, tissue necrosis, reduce shoot
tification of species, subspecies, hybrids, and clones (Castiglione et al., proliferation and rooting (Barampuram et al., 2014). Three surface
1993; Ezzat et al., 2016; Rolland et al., 2016). To our knowledge, this is sterilization methods (1, 2 and 3, Table 2) were compared on D. viscosa
the first molecular study on D. viscosa populations. The outcome of our seeds (about 1000), collected from the three plant populations.
RAPD analysis was highly informative and provided reproducible DNA These methods were based on a mixture of ethanol and sodium
fingerprint profiles for all assayed genotypes. Interestingly, it also re- hypochlorite solutions, at 70% and 1.0%, respectively (methods 1 and
vealed a clear genetic separation among populations, likely associated 2) combined with 0.1% mercury chloride (method 3) and different
to their different geographical origins. Such a marked genetic differ- exposure times. Percentage germination of the sterilized seeds was
entiation probably reflects the diverse environmental conditions in the evaluated ten days after sowing on three different agarized media: MS,
distribution range of this species. Gamborg and Mohr. Methods 1 and 2 resulted highly efficient in killing
Genetic variation within groups of D. viscosa specimens from seeds microorganisms present on the surface of almost all seeds (> 95%),
of the three populations was estimated using different indexes while method 3 dramatically reduced their germination capacity
(Table 1). In general, plants of population C showed a higher level of (< 4.5%) and was, therefore, not suitable. A similar seed surface ster-
intra-genetic variability than those of populations A and B. The AMOVA ilization protocol was reported by Fernandez et al. (2008) in a study
analysis revealed that 23% of the total variation is allocated among finalized to select clones of D. viscosa useful to efficiently remove metals
groups and 77% within groups. The overall Gst and Fst values (0.01 and
0.027, respectively) was different from zero, providing evidence for
Table 2
genetic structuring among groups of plants and populations. Sterilization methods tested on D. viscosa seeds.
This pattern is expected for heterogamous species with a scattered
distribution. The data on genetic structure show that the studied groups Reagents Method 1 Method 2 Method 3
of plants belong to distinct genetic pools, adapted to different en-
Ethanol 70% 30 s 1 min 1 min
vironments. The ecological and edaphic characteristics of their sites of Sterile water 1 min for 5 times 1 min for 5 times 1 min for 5 times
occurrence likely promoted the maintenance of high levels of genetic NaClO solution 1% 5 min 10 min 10 min
variation within native populations. Sterile water 1 min for 5 times 1 min for 5 times 1 min for 5 times
HgCl2 solution 0.1% – – 1 min
Sterile water – – 1 min for 5 times

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F. Guarino et al. Ecotoxicology and Environmental Safety 148 (2018) 675–683

from contaminated soils, also in the presence of mycorrhizal fungi. under a plastic bag, they resulted hardened and able to grow with a
Germination and seedling establishment are key stages in the life cycle high survival rate (96%).
of a plant that often control population dynamics. The germination
percentages of seeds sterilized with methods 1 and 2 were quite similar 3.5. Macroscopic morphological features of the different genotypes
on the three different media (from 7% to 20%), although they varied
among seed batches collected in the different locations. Seeds from Plants obtained from the seeds of populations A and B were quite
population A germinated with a similar percentage (ca. 12%) on all similar in terms of both leaf morphology and bushy habit. During
media, while a low germination rate (> 8%) was recorded for seeds of growth, the stems took on a reddish-brown colour and became woody
population B. Seeds from population C showed the highest germination and tough. On the contrary, plants obtained from the seeds of popula-
rate, reaching 22% on Mohr medium. tion C had elongated leaves and their habit was not bushy, but elon-
Seedlings grew similarly on MS and Gamborg media, while they gated with a limited number of lateral branches and a clear apical
showed a better root development on Mohr medium. On MS medium dominance. Moreover, the growth rate of these plants was very rapid, in
the formation of a large number of shoots longer than 8 cm was ob- particular upwards. The root apparatus of plants from this population
served (data not shown). The importance of macro- and micro-element was more developed than that of the plants of the A and B populations.
composition on seed germination, as well as on seedling development, In fact, the latter genotypes had long roots tending to come out from the
has been reported for several species (Jamshidi et al., 2016; Oberschelp drainage holes of the vessels in which they were growing.
and Goncalves, 2016). This is likely due to the nutritional requirements
for the initial stages of embryo development and optimal plantlet
3.6. Leaf morphology of micropropagated plants
growth.
The morphometric analysis of leaves of micropropagated plants,
3.3. Micropropagation of D. viscosa
acclimatized in the greenhouse, revealed that the leaves of the plants
derived from seeds of population A were elliptic-lanceolate; the leaf
Clonal propagation by microcuttings was extremely effective and
blade length and width were medium. Leaves of the plants obtained
allowed to vegetatively propagate all D. viscosa genotypes. In fact, mi-
from seeds of population B had elliptic-lanceolate/lanceolate leaf
crocuttings adapted well to in-vitro culture and were able to develop a
blades, whose length and width were of medium size. Finally the blade
wide and robust root system 10–12 days after transfer to hormone-free
of leaves of the plants obtained from seeds of population C was gen-
MS medium. The first efficient micropropagation method using nodal
erally lanceolate-shaped, long, and wide.
explants was developed for D. viscosa by Boonne et al. (1992). This
Plants derived from populations A and B had serrated edges, while
protocol proved to be very efficient as more than 10,000 plantlets were
plants from population C showed wavy-toothed edges.
produced in only four months time. Later, Romano (1997) developed a
The morphological data was elaborated by Canonical Variate
cytokinin-induced micropropagation method for this plant using apical
Analysis (CVA; Fig. 2). Component 1 accounted for 94% of the among-
shoot tips as explants. Nodal explants are commonly used for micro-
group variation. The best variables related to component 1 were “leaf
propagation, especially because plants obtained by this technique are
length” and “leaf width”. The two variables “angle of the ribs” and
less prone to genetic variation (Mozafari et al., 2015).
“serrated margins” were related to component 2 only for a low per-
In the procedure described here, we obtained an efficient growth, as
centage (6%). The CVA revealed a good separation among plants de-
well as rooted plantlets of D. viscosa without adding any growth reg-
rived from seeds of populations A and B from those obtained from seeds
ulators and in a relatively short period of time, from five to six months.
of population C. In particular, the two clusters comprising plants from
These results are encouraging in terms of the genetic stability of mi-
populations A and B almost overlapped, suggesting a high similarity.
cropropagated plants since exogenously applied hormones and pro-
Plants from populations A and B were more correlated to the variable
longed culture periods also increase the incidence of genetic variation
“serrated margins”, while plants from population C were significantly
in in-vitro propagated plants (Miguel and Marum, 2011).
correlated with the variables “leaf length” and “leaf width”. Pillai's test
Finally, we chose to adopt MS medium for both seed germination
gave a value of 0.352 indicating statistically significant differences
and micro-propagation because it is easy to use, highly enriched with
macro- and micro-elements and, last but not least, cheaper than the
other tested media.

3.4. Transfer to soil and acclimatization

A very high survival percentage (96%) was obtained during transfer

to soil and subsequent greenhouse acclimatization; no significant dif-
ferences in terms of survival percentage were observed among the di-
verse genotypes of the three collected populations (data not shown). In
fact, most of the plants grew, quickly produced new leaves and were
able to complete their growth cycle. In fact, a critical aspect of in in-
vitro propagation is related to susceptibility to transplantation shocks
leading to high mortality during the final micropropagation stage.
While in in-vitro, plants were continuously exposed to a unique micro-
environment, selected to provide minimal stress and optimal conditions
for survival and multiplication. Thus, their physiological and anato-
mical characteristics must gradually adapt to open-air and soil condi-
tions. Several techniques have been reported to obtain a successful
acclimatization (e.g., lower relative humidity, higher light level, etc.)
and to favour growth performance of micropropagated plants outside Fig. 2. Canonical Variate Analysis (CVA) elaborated on foliar morphological data of
the culture boxes (Hazarika, 2006). In our study, the transferred D. genotypes obtained from seeds of populations A (black), B (red), C (green). (For inter-
viscosa micropropagated plants demonstrated a good capacity to adapt pretation of the references to color in this figure legend, the reader is referred to the web
version of this article.)
to the gardening soil and greenhouse conditions. After an initial period

679
F. Guarino et al.
among analyzed variables (P < 0.001).
It has long been known that the habitat in which a plant grows has
an effect on leaf shape and characters (Royer et al., 2005). The mor-
phometric data was partly congruent with the genetic data related to D.
viscosa populations. Plants deriving from seeds of populations A and B
showed high genetic and morphometric similarities in comparison to
plants of population C. Our seed sampling covered different latitudinal
and longitudinal ranges, but soil features no doubt varied as well
among the three localities. The observed differences (habit, leaf mor-
phology, etc.) among D. viscosa genotypes may be attributed to genetic
factors, but also to the adaptation to diverse environmental conditions.
Moreover, it cannot be excluded that the differences might be due to
epigenetic modifications (i.e., changes in gene methylation level)
caused by the specific environment where the plants were sampled, as
reported by Guarino et al. (2015) for natural populations of Sardinian
white poplar. In general, different environments induce changes in
behaviour, morphology and physiology of plants, collectively named
phenotypic plasticity (Price et al., 2003). Phenotypic plasticity may be
important for adaptation to heterogeneous environments (Jung et al.,
2014; Luo et al., 2016), and it could be interesting for understanding
the processes and environmental features affecting morphology and
intra-specific variability of diverse traits of plants. It is largely docu-
mented that intra-specific variation of morphometric traits influences
species distribution (Zhou et al., 2013), community assembly, eco-
system function (Benomar et al., 2015), and functional diversity (Albert
et al., 2010). Recently, Meng et al. (2017) studied the geographical
variation and the role of climate on leaf traits of Eupteleapleio spermum
Hook f. & Thomson, a relict tree species of China. The authors quan-
tified the intra-specific trait variation along environmental gradients for
predicting species responses to climate changes. This study revealed
that climate was an important driver of leaf trait variation; in fact
49–64% of variation could be explained by combining several climatic
variables.
Our preliminary results showed that D. viscosa plants derived from
seeds of population C could be clearly distinguished from the other
populations, probably because of particular environmental factors, such
as soil features and metal pollution, which characterize the sampled
area. This high variability of leaf traits and, in general, of plant habit
may enhance the ability of D. viscosa to adapt to diverse ecological
niches; however, the interactions between climatic, edaphic, and en-
vironment features and trait variations are complex and, of course,
require additional studies.
3.7. Greenhouse phytoremediation trial
3.7.1. Biomass production
On the basis of our genetic analysis, four of the most biodiverse
genotypes (A17, B6, C3, C17) were assayed for their As phytor-
emediation capacity in a greenhouse pot trial. To this aim, clones of
selected genotypes were grown on soil polluted with 0.5 and 1.0 mg of
As, or on unpolluted soil (CNT). At the end of the experiment, biomass
production and As content in plants and soils were evaluated. Plants did
not show any visual symptoms of toxicity or growth inhibition. The
total dry biomass, measured at the end of the trial, is reported in Fig. 3.
Biomass production depended on the genotype and on the amount
of As added to the soil. In general, the four genotypes produced a si-
milar amount of biomass on unpolluted soil (CNT); only in the case of
clone C17, a significant lower biomass, relative to the other genotypes
grown on unpolluted soil, was recorded.
Biomass production of plants grown on soil contaminated with
0.5 mg of As was inhibited in all genotypes in comparison to control
plants. On the contrary, plants grown in the presence of the higher As
dose (1 mg) produced a greater biomass than those grown with 0.5 mg
As. In the case of clones A17 and B6, biomass production increased in
the presence of the higher As dose even in comparison to plants grown
on CNT soil. By contrast, biomass was inhibited in the presence of 1 mg
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Ecotoxicology and Environmental Safety 148 (2018) 675–683
Fig. 3. Total biomass (dry weight, g) of D. viscosa A17, B6, C3 and C17 clones at the
harvest and for each treatment. Different uppercase letters indicate stastically significant
differences among the biomass produced on the unpolluted soil (CNT) by the four clones
(p < 0.05). Different lowercase (e.g., a, a, a) letters indicate stastically significant dif-
ferences relatively to the same clone (p < 0.05). Bars indicate standard deviations (n =
3).
As in comparison to CNT in clones C3 and C17, although it was slightly
greater than that obtained with 0.5 mg As.
Biomass production was measured separately for leaves, stems and
roots (Fig. 4). In the case of D. viscosa plants grown on CNT soil, organ
biomasses showed, in general, the same trend observed for total bio-
mass in relation to the different genotypes.
On CNT soil, clones B6 and C17 produced the lowest stem and leaf
biomass, respectively. On soil polluted with 0.5 mg As, the lowest root,
leaf and stem biomass was produced by clone B6 and the highest by
A17. In general, clone A17 produced the highest biomass both for
leaves and stems. Root biomass production was lowest for C17, while
the highest was that of A17, confirming its capacity to produce a large
biomass. On soil polluted with 1 mg As, the lowest leaf biomass was
produced by clone C17 and the lowest stem biomass was recorded for
B6. With regard to root biomass, the highest production was measured
for A17. Overall, clone A17 produced the highest leaf, stem, and root
biomass in the presence of As, confirming that the metalloid could
promote, rather than inhibit, growth of this clone. D. viscosa is con-
sidered a pioneer species and its ability to thrive in stressful environ-
ments has been reported (Curadi et al., 2005; Fernandez et al., 2013;
Parolin et al., 2014; Al Hassan et al., 2016). It shows a considerable
capacity to grow on soils that are highly polluted with nickel, magne-
sium, or As (Conesa et al., 2011) and it was also proposed as bio-in-
dicator plant for these HMs and Ms (Ater et al., 2000). However,
Fernandez et al. (2013) reported that Cd induced visible symptoms of
phytotoxicity, such as reduced growth, at concentrations higher than
5.0 mg L−1 for the assayed genotypes. In our study, As led to a lower
biomass production at the lower As dose (0.5 mg) relative to controls,
but a greater biomass was produced in the presence of the higher As
dose in comparison with the lower one. In the case of clones A17 and
B6, a stimulatory effect on biomass was induced by As when compared
with controls. A similar result was reported for the hyperaccumulator
plant Pteris vittata L. where As soil enrichment significantly increased
frond biomass, even though it slightly decreased root biomass (Das
et al., 2017). On the contrary, present results show that the highest
biomass production was recorded for roots of D. viscosa, in particular in
the case of clone A17. This stimulating effect at low As concentrations
was observed by several authors ( Woolson et al., 1973; Carbonell-
Barrachina et al., 1998; Miteva, 2002; Garg and Singla, 2011). The fact
that this phenomena occurs under axenic conditions in cultured plants,
such as Arabidopsis thaliana (Chen et al., 2010), indicates that the trait is
not based on As disrupting plant-biotic interactions. Instead, it results
either from a direct interaction of As with plant metabolism, or from an
F. Guarino et al.
Fig. 4. Biomass (dry weight, g) of D. viscosa leaves, roots and stems of clones A17, B6, C3
and C17 at harvest and for each treatment. Different uppercase letters indicate statisti-
cally significant differences among the biomass produced on unpolluted soil (CNT) for
each organ among the clones (p < 0.05). Different lowercase letters (e.g., a, a) indicate
statistically significant differences within clones and treatments respect to the same organ
(p < 0.001). Bars indicate standard deviations (n = 3).
interaction of As with plant nutrients (Finnegan and Chen, 2012). While
the mechanism is unknown, it has been suggested that the growth
benefit arises from As stimulation of phosphate uptake (Tu and Ma,
2003).
3.7.2. Arsenic accumulation in plants and soil reclamation
At the start and at the end of the greenhouse trial, As concentration
was below detection limit (< 2.5 μg g−1), in all analyzed soils. The As
volatilization due to soil microorganism and climatic greenhouse con-
ditions was less than 15% of the total amount. All genotypes took up As
and translocated it to the leaves (Fig. 5).
Thus, the metalloid was detectable in leaves, but not in the other
plant organs. The extent of As accumulation in leaves was genotype-
dependent. In fact, As concentration in each analyzed clone was highly
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Ecotoxicology and Environmental Safety 148 (2018) 675–683
Fig. 5. Arsenic concentrations in leaves of D. viscosa clones A17, B6, C3 and C17 at the
harvest and for each treatment. Different letters indicate significantly different values for
treatments (p < 0.001). Bars indicate standard deviations (n = 3; l.o.d. = limit of de-
tection).
variable and differences were statistically significant (p < 0.001).
Clone A17 was able to accumulate the highest amount of As
(60 μg g−1), followed by C3, B6, and C17 clones. In A17, leaf As con-
centration was 4- to 30-fold higher than in leaves of C3 and C17 clones,
respectively.
In general, plants grown on soil contaminated with 0.5 mg As
showed the highest As concentration when compared to those grown on
1 mg As-contaminated soil; in the case of A17, there was no difference
either in the presence of 0.5 or 1 mg As. In the case of clone B6, the
highest As concentration was observed with 1.0 mg As treatment.
However, the plants of this clone showed a lower leaf biomass pro-
duction relative to controls both in the presence of 0.5 and 1 mg As. In
general, clone A17 showed the best performance with respect to As
accumulation also in the presence of 1 mg As. This clone was able to
accumulate in the leaves ca. 44.0 and 37.2 μg of As in the presence of
0.5 and 1.0 mg As, respectively. Given that As was detected only in the
leaves, this value corresponded to the total As amount that A17 clone
was able to accumulate; thus, the amount taken up corresponded to
about 9% and 4% of As doses added to the soil (0.5 and 1 mg, re-
spectively). A good performance in terms of As accumulation was also
obtained with clone C3; in fact, the As content in C3 leaves was equal to
8.6 and 5.2 μg (0.5 and 1.0 mg As, respectively), representing 1.7% and
0.5% of the initial amount of As added to the soil. The other two clones,
B6 and C17 accumulated only a very low amount of As (i.e., 1.6 and
0.6 μg in the presence of 1 mg and 0.5 mg As, respectively). Therefore,
these two clones accumulated only 0.16% and 0.11% of the initial
content of As in the soil. Taking into account the total amount of As
initially added to the soil, the percentage of As volatilization from soil
without plant, the amount of As estimated in the plant biomass and in
the soils at the end of experimentation, we estimated a removal effi-
ciency ranging from 75% to 80%, depending on the total amount of the
contaminant added to the soil (0.5 or 1 mg As) and also on clone (A17
or C3).
The capability of D. viscosa species to take up As has been reported;
this plant showed the highest translocation factor (TF- i.e., the ratio
between concentration in the aerial organs relative to the roots) for
many HMs and Ms (Conesa et al., 2011). Similar to our results,
Gonzalez-Fernandez et al. (2011) obtained TF values of 2 for Zn and 1.5
for Pb in D. viscosa grown on wastes from a nearby mining area. In our
case, the TF cannot be estimated because root As concentration was
below detection limit. From a mathematical point of view, the ratio of a
positive value (e.g., As leaf concentration) with an arbitrarily small
positive quantity (e.g., As root concentration) is a very large number
(> > 1); therefore, we believe that these clones of D. viscosa can be
F. Guarino et al.
considered good As accumulators, as proposed by Mattina et al. (2003).
A similar pattern of As accumulation was observed by Pistelli et al.
(2017) in the case of a mining area of Elba Island (Italy). In that study,
D. viscosa significantly accumulated As, translocating it to the aerial
organs according to the following pattern: leaves > stems > roots; a
similar trend was also observed by Baroni et al. (2004).
Since, at the end of the trial, As was not detectable in the soil, we
can assume that all D. viscosa clones took up the metalloid, translocated
it to the leaves, and that, from there, it was volatilized into the air. This
mechanism was proposed and demonstrated for other plant species
(Doucleff and Terry, 2002; Mirza et al., 2011). Some authors also hy-
pothesized that As is absorbed as arsenate As(V) and a part of it is
converted into arsenite [As(III)] by arsenate reductase (Finnegan and
Chen, 2012). Arsenite is transferred and stored in the vacuole, later
mobilized in the cell cytosol, moved into the air spaces of the leaf and
finally volatilized in the air. In our study, the soil was polluted with
sodium arsenite (NaAsO2), a form that is completely available for the
plant. As(III) is uncharged at neutral pH in flooded paddy soils and is
more soluble and mobile compared to As(V) (Ji et al., 2017). Recent
studies reported that in rice, As(III) can enter into cells mainly through
nodulin 26-like intrinsic proteins (NIPs), a subfamily of aquaporin
proteins that belong to the major intrinsic proteins (MIPs), a gene fa-
mily which essentially facilitates the diffusion of water and small un-
charged solutes in all life domains (Ali et al., 2009; Li et al., 2016;
Chauhan et al., 2017).
4. Conclusions
The experimentation we performed describes efficient protocols for
both seed germination and the in vitro bud multiplication of D. viscosa.
Morphological and molecular analyses revealed that D. viscosa clones of
the different analyzed populations were morphologically and geneti-
cally diverse in relation to geographical origins. Then, the developed
protocol could be efficiently used to sustain the propagation and con-
servation of selected clones of this species, useful for phytoremediation
purposes. The experimentation on As-polluted soils revealed diverse
phytoremediation performances of different clones of D. viscosa (A17,
B6 and C17). In particular, although B6 and C17 clones produced a
lower biomass respect to clone A17, they were able to reclaim the
contaminated soils while keeping a very low As amount in their organs.
Therefore, these selected D. viscosa clones might be potentially used for
phytoremediation of As-polluted soils via an efficient phytovolatiliza-
tion process. They would decrease the risks of biomagnification and
reduce the amount of contaminated biomass to dispose after phytor-
emediation, while providing environmental and economic benefits as
compared to traditional soil reclamation procedures.
Acknowledgements
Thanks to Prof. Stefania Biondi for English revision of the text. The
research was supported by funds for didactic activity in “Applied
Biology and Biodiversity” by the University of Salerno
The authors have declared that no competing interests exist.
Appendix A. Supporting information
Supplementary data associated with this article can be found in the
online version at http://dx.doi.org/10.1016/j.ecoenv.2017.11.010
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