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Nanoparticles Effects On Growth and Differentiation in Cell Culture of Carrot (Daucus Carota L.)
Nanoparticles Effects On Growth and Differentiation in Cell Culture of Carrot (Daucus Carota L.)
Nanoparticles Effects On Growth and Differentiation in Cell Culture of Carrot (Daucus Carota L.)
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Received 00 Xxxxxx 2010 – Received in revised form 00 Xxxx 2010 – Accepted 00 Xxxxx 2010
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.
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.
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.
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