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Nanoparticles effects on growth and

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Agrochimica, Vol. LIV - N. 6 November-December 2010

Nanoparticles effects on growth and differentiation in cell cul-

ture of carrot (Daucus carota L.)
L. GIORGETTI1*, M. RUFFINI CASTIGLIONE2, M. BERNABINI1, C. GERI1
1
Institute of Agricultural Biology and Biotechnology (IBBA/CNR), UOS Pisa, Pisa, Italy
2
Department of Biology, University of Pisa, Pisa, Italy

Received 00 Xxxxxx 2010 – Received in revised form 00 Xxxx 2010 – Accepted 00 Xxxxx 2010

Keywords: carrot in vitro culture, mitotic activity, nanoparticles, somatic

embryogenesis

Introduction. – In the last decade the development of nanoscienc-

es and nanotechnologies have offered their great potential in introduc-
ing new materials, improving the quality of life and in creating novel
knowledge-based sustainable processes. Undoubtedly nanotechnologies
have provoked the proliferation of new industrial activities involving the
production and use of nanoparticles (NPs), different for chemical com-
position, surface treatment, size and shape. In this scenario, it becomes
urgent the need of a risk assessment associated to these new entities for
animal/human health and for the environment. The impact of engineered
NPs on growth, development and functions of plants in general, those
of agronomic and alimentary interest, as well as model plants, when
exposed in chronic and cumulative fashion to NPs present in soil/water,
need to be assessed. NPs are defined as such if their size ranges between
1-100 nanometres. They acquire new physicochemical properties, differ-
ent from the bulk material. Literature presents reports related to adverse
effects of some NPs on living organisms. In case of higher animals,
NPs exposure due to environmental pollution from natural or industrial
processes, particularly in case of aerosol NPs that are easily inhaled, can
induce serious health hazards like pulmonary inflammation and cancer.
NPs, because their small size, can easily enter in vital organs as bone
marrow, lymph nodes, nervous system, spleen and heart, inducing seri-
ous pathologies (Gonzales et al., 2008; Oberdörster, et al., 2005,
Gianutsos et al., 1997; Gibaud et al., 1994; Gibaud et al., 1996).
As far as plants are concerned, studies on the topic are quite recent
and not very extensive. The researches are based on the investigation of
NPs accumulation and morphological/physiological parameters altera-

*Corresponding author: l.giorgetti@ibba.cnr.it

 L. giorgetti et al.

tions (seed germination, root growth and elongation) on different higher

plant species like radish, rape, ryegrass, lettuce, corn and cucumber,
after in vivo treatments with various NPs as alumina (Yang et al.,
2005), magnetite (Zhu et al., 2008), aluminium, zinc and zinc oxide and
multi-walled carbon nanotube (Lin and Xing 2007). Toxic responses
on seed germination and root elongation greatly depend on the chemical
nature of the tested NPs and on their concentration. Moreover since the
NPs negative effects are detectable only in some of the considered plant
species, the root system morphology and the diverse capacity to adsorb
nutrients can play a key role in the toxic response as suggested by dif-
ferent authors (Khodakovskaya et al., 2009; Ma et al., 2010; Lee et
al., 2010).
Moreover no effects of Titanium oxide NPs were reported on adult
willow plants (Seeger et al., 2009), whilst Palladium NPs can affect
pollen vitality in kiwifruit plants (Speranza et al., 2010). Recently
cytological studies demonstrated mitotic activity inhibition and cyto-
logical aberrations in maize root tip apices exposed to magnetic NPs
(Racuciu et al., 2009), in silver NPs treated Allium cepa root apices
(Kumari et al., 2009) and in root tips of Vicia narbonensis and Zea
mays treated with TiO2-NPs (Ruffini Castiglione et al., 2010).
At the moment few studies have been performed on plant cells in
culture (Lin et al., 2009; Tan et al., 2009), while animal cell culture has
been one of the main tools in this sort of research.
In this work the effects of NPs were investigated exploiting the in
vitro somatic embryogenesis of Daucus carota L. Somatic embryogen-
esis is a well suitable system for the analyses of the differentiation proc-
ess in plants in standard and in stress conditions. In contrast to zygotic
embryogenesis, somatic embryogenesis can easily be observed, the
external conditions of the embryos can be experimentally controlled and
large quantities of embryos can by far be obtained. Since the first reports
of somatic embryogenesis (Reinert, 1958; Steward et al., 1958),
carrot has been used as a model plant for cellular, physiological and
molecular study. Generally carrot cells are mantained in a medium that
contains auxin and proliferates in an undifferentiated manner. Somatic
embryogenesis is easily obtained by removal of auxin from the medium
at which point subsequent differentiation to globular heart-shaped and
torpedo- shaped embryos occurs.
In this research the model system of somatic embryogenesis of
Daucus carota L. was utilized in order to check Fe3O4 NPs effects on
 titolo breve 

developmental processes like cell growth, de-differentiation, and somat-

ic embryogenesis (Nuti Ronchi and Giorgetti, 1995; Giorgetti et
al., 1995; Geri et al., 1999). Fe3O4 NPs (magnetite) were chosen for
their structural stability and non-toxicity after accumulation in plant tis-
sues in vivo.

Materials and methods. – Seed sterilization. – Carrot seeds (Daucus carota L.,
cv. Berlicum) were surface sterilized with 3% sodium hypoclorite for 30 min and then
rinsed several times with sterile distilled water. Seeds were germinated in hormone-free
MS (Murashige and Skoog, 1962) solid medium (0.8% agar).
Cell culture. – Carrot cultures were set up from 2 to 3-mm sections of 2-cm
hypocotyl explants of 1-week germinated seeds. These were maintained in liquid medi-
um, MS supplemented with 2.2 μM 2,4-dichlorophenoxyacetic acid (2,4-D) (MS+) for
20 days under continuous light (6–12 μmol•m-2•s-1) at 24°C with agitation at 80 rpm,
as described by de Vries, et al. (1988). Cell lines were filtered away from the hypocotyl
cultures via 400-μm pore size nylon sieves and subcultured in the above medium.
Embryogenesis induction. – To induce embryogenesis, the 7-day-old cell line sub-
cultures were filtered first through a 120-μm and then a 50-μm pore size nylon sieve.
The cell clumps, referred to as cellular units (CU), which were collected by the second
filter, were washed several times with hormone-free MS medium (MS-), and grown
without agitation in Petri dishes at a resuspension density of 3000 CU/mL in 5 ml total
MS- volume. Cells were counted using Nageotte counting chamber.
Nanoparticles treatments on cell cultures. – Fe3O4 NPs, 6nm diameter, were
dispensed at doses of 2.01 mg/l, 4.02 mg/l, 6.70 mg/l, 20.10 mg/l e 33.5 mg/l in MS+
medium during indifferentiate cell growth, starting from the first day of subculture. The
effects of Fe3O4 NPs on cell growth were determined by measuring the volume of packed
cell sediment at different times during the 14 days of subculture.
Nanoparticles treatments on embryogenic differentiation:
a) Pre-treatment with NPs during undifferentiated cell growth: carrot cell cultures
were grown for 7 days in MS+ with doses 20.10 mg/l and 33.5 mg/l before inducing
somatic embryogenesis in MS-, NPs free medium.
b) Treatment with NPs during somatic embryogenesis: carrot cell cultures were grown
for 7 days in MS+, then cells were induced to differentiate somatic embryos in MS-
in presence of NPs 20.10 mg/l and 33.5 mg/l.
As controls, embryos from not treated cell cultures have been estimated. Since
Fe3O4 NPs were suspended in a solution of 0.5 mM tetramethylammonium hydroxide
(TMAOH), parallel experiments were carried out on cells cultures grown in presence of
TMAOH solvent as further control. To analyze the efficiency of somatic embryogen-
esis obtained in the different experimental condition, somatic embryos were verified
after 10 days from induction. Five Petri dishes for each treatment, with initial density
3000 CU/mL in 5 ml total volume, were counted at the stereomicroscope. The collected
data were statistically elaborated by Anova and with post-hoc Bonferroni test for mul-
tiple comparisons.
Cytological analysis and Mitotic index determination. – 5 ml of carrot cells suspen-
sion culture grown in MS+, were centrifuged to collect cells that were fixed for 24h in
Carnoy’s fixative (ethanol 100%: acetic acid; 3:1) at 4 days of culture. Then fixative was
 L. giorgetti et al.

washed and the material was stained with Feulgen’s staining protocol for cytological
analysis (Giorgetti et al., 1995). Mitotic index (MI) was calculated by counting the
number of mitoses in 1000 cells. 5 different slides were analysed for every treatment.
The collected data were statistically elaborated by Anova and with post-hoc Bonferroni
test for multiple comparisons.

Results. – The effects of Fe3O4 (magnetite) NPs treatments on

carrot cell cultures were analyzed considering three different param-
eters: NPs response during undifferentiate cell growth, NPs response
during the acquisition of embryogenic potential and finally NPs effects
throughout somatic embryogenesis.
Growth curves trend, determined by measuring the volume of packed
cell sediment at different culture times, clearly showed that Fe3O4 NPs
exposure from 2.01 to 6.7 mg/l did not significantly influence growth,
while 20.10 mg/l concentration greatly affected cell proliferation in
carrot cultures that stopped growing totally at the dose of 33.5 mg/l
(Fig. 1).
These results were partly in agreement with mitotic index (MI)
analysis (Fig. 2): mitotic activity was moderately reduced at the lower
concentrations of Fe3O4 NPs (MI 5.2% in the control, it decreased to
3.1% in 2.01 mg/l, 3.2% in 4.02 mg/l, and 2.5% in 6.7 mg/l of Fe3O4
NPs). Also treatments with
0.5 mM TMAOH solvent
alone (which had no effects
on growth curve) had a nega-
tive outcome on MI (3.2%).
At 20.10 mg/l of Fe3O4
NPs, mitotic index dropped
dramatically (MI 0.8% in
20.10 mg/l Fe3O4 NPs), and no
mitotic divisions were present
at the dose of 33.5 mg/l, in
accordance with growth curve
results (Fig. 1 and 2).
To test the possible effect
of NPs on somatic embryo-
Fig. 1. – Carrot cell growth in cultures after treatment
with different concentrations of Fe O NPs 6 nm diameter;
3 4
genesis, carrot cell cultured
a) control cell growth in MS+ compared with cell growth for the first 7 days in MS+
in MS+ and TMAOH solvent; b), c), d), e), f) comparison
between control and cell growth in MS+ added with Fe O
3 4
were induced to differentiate
NPs 2.01 mg/l, 4.02 mg/l, 6.7 mg/l, 20.10 mg/l and 33.50 somatic embryos (Fig. 4), by
mg/l, respectively.
 titolo breve 

removing both the hormone

and the NPs. Only the higher
concentration of NPs (20.1
and 33.5 mg/l) that showed
effect on cell growth were
considered. Surprisingly car-
rot cells were able to recov-
er embryogenic capacity in
fresh MS- without NPs as
they started to differentiate Fig. 2. – Effect of Fe O NPs 6 nm diameter on mitotic
3 4

somatic embryos but at a index in carrot cell cultures after 4 day in MS+. Different
concentrations of Fe O NPs (2.01 mg/l; 4.02 mg/l;
lower level in comparison 6.7 mg/l; 20.10 mg/l; 33.50 mg/l) were tested. Mitoses
3 4

to the control untreated cells detected on a total of 5000 cells. Asterisks indicate sig-
(Fig. 5). Carrot control cells nificant differences from the control (p< 0.05).
differentiated an average of
520 somatic embryos (on a total of 15000 UC) while TMAOH, 20.2
mg/l and 30.5 mg/l Fe3O4 NPs treatments differentiated 410, 420 and
340 somatic embryos respectively. Only the difference between the
control and the higher NPs concentration was statistically significant.
On the contrary, when carrot cells grown in MS+ were differentiated
in MS- in the presence of NPs, cells were blocked and no somatic
embryos were later on obtained (Fig. 6); moreover also 0.5 mM
TMAOH treatment affected somatic embryogenesis and the obtained
embryos were greatly reduced (from a total of 520 embryos per 5 ml
in the control to 100 embryos in TMAOH treatment in the same vol-
ume).

Fig. 3. – Carrot cells in culture analyzed after fixation and Feulgen’s staining to determine mitotic index.
Fig. 3a) control cells with mitoses (MI = 5.2%); Fig. 3b) carrot cell culture treated with 20.1 mg/l Fe3O4
NPs in which mitoses are not present. In this case the determined MI was very low (MI = 0.8%, see fig. 2);
bar = 10µm.
 L. giorgetti et al.

Fig. 4. – Developmental stages of carrot somatic embryos; from left to right: globular, heart, torpedo and
plantula stage.

Discussion. – The data

presented above clearly indi-
cate that the in vitro model
system of somatic embryo-
genesis in Daucus carota L.
can be successfully used to
check the impact of Fe3O4
NPs both during undiffer-
entiated growth as well as
Fig. 5. – Effect of Fe3O4 NPs 6 nm diameter on somat- the various steps of somatic
ic embryogenesis in carrot cell cultures. Fe3O4 NPs
20.10 mg/l and 33.50 mg/l were given for 7 days during embryogenesis. In vitro undif-
the undifferentiated growth in MS+, then cells were ferentiated cell cultures were
induced to somatic embryogenesis in MS- (without hor-
mone and Fe3O4 NPs). Somatic embryos were counted
analysed considering various
after 10 days from embryogenesis induction, on a total of parameters, as growth curve,
15000 CU. Asterisks indicate significant differences from
the control (p< 0.05).
mitotic index and a thor-
ough cytogenetic analysis,
which allowed quantifying
the effects of NPs exposure.
Particularly important was
the examination around the
3th-5th day of culture in fresh
MS+ (which represents the
beginning of the logarithmic
growth). This time was the
best suited to detect likely
mitotic index anomalies; the
Fig. 6. – Effect of Fe3O4 NPs 6 nm diameter on somatic
embryogenesis in carrot cell cultures. Treatments with
obtained results indicated that
Fe3O4 NPs 20.10 mg/l and 33.50 mg/l were done starting Fe3O4 NPs treatments affected
from somatic embryogenesis induction in MS-. Somatic significantly MI also at the
embryos were counted after 10 days from embryogenesis
induction on a total of 15000 CU. Asterisks indicate sig- lower doses and, since no dif-
nificant differences from the control (p<0.05). ferences were found in the
 titolo breve 

relative growth curves, probably NPs treatments can influence cell vol-
ume also. In fact cells undergoing mitosis are meristematic cells that are
easily recognized for their small size; later on these cells considerably
enlarge and stop dividing.
Our results were in agreement with very recent findings (Santos
et al., 2010) carried on suspension cultures of Medicago sativa in wich
a cell biology approach was used to evaluate the impact of Quantum
Dots (QDs) nanocrystals on in vitro plant cells; here it was shown the
toxic effect of QDs on cell growth and cell viability caused by oxidative
stress and ROS accumulation. In addition same results were obtained in
in vitro cultured cell of Arabidopsis thaliana exposed to multi-walled
carbon nanotubes (Lin et al., 2009). Up to now, the analyses of NPs
effects on plant development was limited to the processes of seed ger-
mination and root elongation (Ruffini Castiglione et al., 2010; Lin et
al., 2007; Ma et al., 2010) but no additional studies were performed in
in vivo or in in vitro plant systems to test the effect of NPs during plant
embryogenesis. To the best of our knowledge this is the first report on
the effects of nanoparticles on plant embryogenesis.
In this work we examined the effects of Fe3O4 NPs throughout carrot
somatic embryogenesis by considering the efficiency of differentiation
steps and somatic embryos production. It is well known from the literature
on carrot somatic embryogenesis (de Vries et al., 1988; Giorgetti et
al., 1995) that cultured carrot cells acquire embryogenic capacity during
the first 7 days of culture in MS+; at this time PEMs (pro-embryogenic
masses) and single embryogenic cell are already formed from mitotically
active cells. When the cultures were treated with Fe3O4 NPs at higher con-
centrations (20.1 and 30.5 mg/l) for the first 7 days in MS+ (before embry-
ogenesis), MI was radically lowered although, if NPs were removed, cells
could totally recover and start the differentiation process towards somatic
embryos formation. NPs treatments really represented severe stress condi-
tion for the undifferentiated cell culture; however it was ascertained that
stress-related plant reactions (Zavattieri et al., 2010) can induce and
enhance embryogenic capability in in vitro cultured cells.
On the contrary when embryogenic carrot cells with evident PEMs
and single embryogenic cells were induced to differentiate in the pres-
ence of Fe3O4 NPs, no embryos formation was observed since lasting
stress condition directly blocked the embryogenetic pathway.
Then our data demonstrated specific effects of Fe3O4 NPs, more
remarkable on developmental processes than on proliferative growth in
in vitro carrot cell system.
 L. giorgetti et al.

NPs impact on developmental processes, as suggested by these

results, can pose a claim on crops yields, due to possible widespread
pollution, as well prompting the hint of yet unknown mode of action.

Acknowledgment. – We thank Dr. Diana Boraschi (ITB, CNR,

Pisa) for kindly provided us Fe3O4 NPs and the relative solvent.
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Abstract. – Nanoparticles (NPs) effects on developmental processes like cell

growth, de-differentiation, and embryogenesis were analyzed using the in vitro model
system of Daucus carota L. somatic embryogenesis. Fe3O4 NPs, 6 nm diameter, were
dispensed at doses ranging from 2.01 to 33.5 mg/l to carrot in vitro cultures at different
times, along the stages of cell growth and embryogenic differentiation. Moreover, the
mitotic index of cell cultures under treatments has been cytologically determined. The
results demonstrated that Fe3O4 NPs exposure from 2.01 to 6.70 mg/l slightly affected
growth, mitotic index, and de-differentiation. At the dose of 20.10 mg/l cell growth
and relative mitotic index dropped dramatically, and stopped completely at 33.5 mg/l.
Moreover, these higher doses, entirely blocked embryo formation when treatments were
done along somatic embryogenesis induction. Therefore, this model system seems well
suited to check NPs impact on developmental processes.

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