Agrochimica, Vol. LIV - N. 6
November-December 2010
Nanoparticles effects on growth and differentiation in cell culture of carrot (Daucus carota L.) L. GIORGETTI1*, M. RUFFINI CASTIGLIONE2, M. BERNABINI1, C. GERI1 1 2
Institute of Agricultural Biology and Biotechnology (IBBA/CNR), UOS Pisa, Pisa, Italy 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 nanosciences and nanotechnologies have offered their great potential in introducing 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 composition, 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, different 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 serious 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:
[email protected]
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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 different 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 cytological 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 embryogenesis is a well suitable system for the analyses of the differentiation process 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
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developmental processes like cell growth, de-differentiation, and somatic 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 tissues 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 medium, 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 subcultures 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 MSin 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 embryogenesis 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 multiple comparisons. Cytological analysis and Mitotic index determination. – 5 ml of carrot cells suspension 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
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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 parameters: 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 negative 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 embryoFig. 1. – Carrot cell growth in cultures after treatment genesis, carrot cell cultured with different concentrations of Fe O NPs 6 nm diameter; 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 were induced to differentiate between control and cell growth in MS+ added with Fe O 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 3
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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 carrot cells were able to recover embryogenic capacity in fresh MS- without NPs as they started to differentiate Fig. 2. – Effect of Fe O NPs 6 nm diameter on mitotic 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 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 volume). 3
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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.
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Fig. 4. – Developmental stages of carrot somatic embryos; from left to right: globular, heart, torpedo and plantula stage.
Fig. 5. – Effect of Fe3O4 NPs 6 nm diameter on somatic embryogenesis in carrot cell cultures. Fe3O4 NPs 20.10 mg/l and 33.50 mg/l were given for 7 days during the undifferentiated growth in MS+, then cells were induced to somatic embryogenesis in MS- (without hormone and Fe3O4 NPs). Somatic embryos were counted after 10 days from embryogenesis induction, on a total of 15000 CU. Asterisks indicate significant differences from the control (p
