ASSESSMENT OF METAL NANOPARTICLE-INDUCED MORPHOLOGICAL AND PHYSIOLOGICAL CHANGES IN IN VITRO CULTURES OF RAPESEED (BRASSICA NAPUS L.)
DOI:10.30825/5.EJPAU.166.2018.21.4

Magdalena Tomaszewska-Sowa¹, Anetta Siwik-Ziomek², Anna Figas¹, Karol Bocian^1
1 Department of Agricultural Biotechnology, University of Science and Technology, Bydgoszcz, Poland
² Department of Biogeochemistry and Soil Science, University of Science and Technology, Bydgoszcz, Poland

ABSTRACT

To understand the mechanisms involved in plant responses to metal nanoparticles, the morphological and physiological changes in rapeseed plants (Brassica napus L.) were determined. The sterile seeds were inoculated onto the MS medium (sterilized and unsterilized) containing silver, copper and gold nanoparticles at the concentration of 20 ppm. Silver nanoparticled did not inhibit the germination process as opposed to cooper nanoparticles. The nanoparticles did not affect the roots length. The seedlings grown on the medium supplemented with nanoparticles were significantly shorter in comparison to the control. Nanosilver without autoclave sterilization reduced the plant height. The total chlorophyll, the ratio of chl a / chl b and carotenoid content decreased after the exposure to metal nanoparticles. Additionally, it was found that the nanoparticles of gold increased the activity of catalase, as compared with the control. There is a growing number of research of the biological effects of nanoparticles on higher plants. The application of nanoparticles in in vitro cultures can be very important to facilitate understanding the nanoparticles toxicity for plants.

Key words: catalase, chlorophyll, nanocopper, nanogold, nanosilver, oxidative stress.

INTRODUCTION

Plant growth and development are controlled by internal regulators that respond to environmental conditions. Under sub-natural conditions the plants show perturbations in their biochemical and physiological processes. The factors affecting the condition of plants include metal nanoparticles (NPs). NPs may pose a toxicological risk as nanosized particles have presented toxicity in a variety of organisms being generally more harmful than large particles [30]. Due to its specific structure and differences from metallic, nanometals can demonstrate a number of interesting features [28, 42]. The fields in which the properties of nanosilver are emphasized as well as which have supported numerous research include medicine, pharmacy, biotechnology, cosmetology, food and construction industry [6, 8, 33]. However, the environmental impact of silver nanoparticles (AgNPs) remains uncertain [45]. AgNPs and other metal nanoparticles, like nanocopper (CuNPs) or nanogold (AuNPs) are used widely as antimicrobial and antifungal nanomaterials. Nanosilver shows a strong antibacterial and antifungal effect, stronger than any other metal nanoparticles [2]. As Abdi and coworkers reports nanosilver used for disinfection of Valeriana officinalis L. single node explants in in vitro cultures, was the most successful disinfection treatment. Nanosilver had significant differences with other treatments [2]. Fakhrfeshani et al. [9] report that the 200 mg kg^-1 nanosilver solution applied after proper sterilization, had successfully controlled bacterial and fungal contamination in gerbera tissue culture. Despite the advantages, all metals are toxic at higher concentrations due to oxidative stress by the formation of free radicals. Another reason why metals may be toxic is that they can replace essential metals in pigments or enzymes disrupting their function [11]. The interaction between plants and nanomaterials is still under study. Most research cover silver nanoparticles on plants has been still in progress [17, 27]. There exist different and often conflicting reports on the absorption, translocation, accumulation, biotransformation, and toxicity of NPs on various plant species [16, 39]. The toxicity of AgNPs has been demonstrated in various prokaryotic organisms and mammalian cell lines [23]. The effect of AgNPs on land plants seems to be dependent on the age and plants species, development stage, various experimental conditions and the method and time  of experimental exposure. In land plants, a different and conflicting effect of AgNPs has been reported, depending on various nanoparticle properties, for example size, shape, surface coating or the aggregation state [45]. Under changed environmental conditions (metal nanoparticles-NPs), plants demonstrate differences in their biochemical and physiological processes. The detailed mechanism and toxic effect of nanoparticles are not completely understood. The research has shown AgNPs shed ionic silver into surrounding and cause oxidative stress and inhibition of respiratory enzymes by generating reactive oxygen species [38, 40]. There are also some hypotheses, including cell wall damage and DNA/protein damage [19]. The most common phytotoxic effect is associated with NPs stress which can increase production and detoxification of reactive oxygen species (ROS) determining the factor of oxidative stress and cell damage [24]. Redox reactions, involving electron exchange, enzymatic response, are among the biochemical processes affected by external environment changes. Therefore, even under seemingly favorable environmental conditions, a plant continuously produces ROS. The ROS accumulation is potentially harmful for the growth and development of plants. Thus the occurrence of oxidative stress, through the generation of free radicals, is an inevitable by-product of a normal plant metabolism. ROS are continuously reduced and detoxified by an extensive antioxidant system. Nanoparticles are a well-known cause of oxidative stress and they can activate the plant antistress defense mechanism. The research has shown that NPs smaller than 100 nm in size can modify the morphological and physiological properties. A good marker of oxidative stress is the chlorophyll content, the activity of antioxidant enzymes and growth parameters. The aim of this study has been to determine the effect of the presence of metal nanoparticles in the medium on the growth and development of rapeseed (Brassica napus L.) as well as on the chlorophyll content and the activity of antioxidant enzyme, catalase, in seedlings from in vitro cultures.

MATERIALS AND METHODS

Nanoparticles
During the experiment, following freshly prepared metal nanoparticles were used as the sterilizing agent added to the medium: nanosilver, nanocopper and nanogold in water suspension provided by the Department of Biogeochemistry and Soil Science of the UTP University of Science and Technology in Bydgoszcz. The nanoparticles suspension (the nanoparticle size from 30 nm ± 5 nm) is stabilized by PVP, water soluble polymer preventing particles aggregation.

Plant material
The research material was made up of the seeds of spring rapeseed (Brassica napus L.  ‘Margo’ cultivar). In the experiment a standard sterilization method was applied. The seeds were pre-sterilized in 70% ethanol for 60 seconds, then sterilized in 30% ACE (commercial bleach, containing 5% sodium hypochlorite) for 8 minutes. The rape seeds were rinsed in sterile bidistilled water three times for the sterilizing agent to be removed. The sterilized seeds were inoculated onto sterilized and unsterilized medium prepared compliant with different variants provided in Table 1. The medium used in experiment [25] was diluted in the ratio 1:2 and solidified with 0.7% Vitro Lab-Agar (Biocorp); it was brought up to pH 5.6–5.8. In the first variant of the experiment, rape seeds were inoculated onto the sterilizing ½ MS medium containing metal nanoparticles at the concentration of 20 ppm. Tissue culture media were sterilized by autoclaving at 121°C and 1.5 kg cm^-2 for 20 minutes. In another variant, rape seeds were inoculated onto unsterilized ½ MS medium containing metal nanoparticles at the above concentration (Tab. 1). ½ MS sterile medium without nanoparticles was used as the control in both variants of the experiment. The experiment was carried out in glass jars, using 50 ml of the nutrient medium each time. The germination process and shoot growth occurred under controlled conditions: a 16-hour photoperiod, the light intensity of 40 µmol·m^-2·s^-1, temperature of 25 ± 2°C, and air humidity of 70%. The experiment was performed in three replications, 75 seeds for each variant. After two weeks, the surface contamination was visually evaluated and the germination percentage was calculated (the ratio of the number of seeds germinated to the total number of seeds). After next two weeks the root and shoot length were measured. In addition, the chlorophyll and carotenoid contents and catalase activity in the plant extract were determined.

Estimation of chlorophyll and carotenoid contents
Chlorophylls were extracted from 100 mg leaves, with 10 ml of concentrated 80% acetone. Absorbance was measured for chlorophyll a and b; 645 and 663 nm, respectively. Carotenoids were extracted with 100 mg of concentrated 80% acetone (10 ml). The absorbance was measured at the wavelength of 440 nm for carotenoids. The concentration of chlorophyll a and b and total carotenoids was calculated according to Wettstein [43].

Enzyme extraction and Assay
In order to determine the activity of catalase, shoots growing in in vitro conditions were homogenized with suitably cooled 0,067 M phosphate buffer (KH₂PO₄, Na₂HPO₄) pH 7,0. The homogenates were centrifuged and the supernatants were used to determine the activity of catalase. The assay mixture for the catalase activity comprised 1.25·10^-2 M H₂O₂, diluted enzyme extracted and 0,067 M phosphate buffer. Catalase activity was spectrophotometrically determined according to Lück [20]. The determination consisted in measuring the decrease in ultraviolet light absorption during the decomposition of H₂O₂ within 60s by catalase at the wavelength of  λ=240 nm and activity expressed as µmol H₂O₂ min^-1 g^-1 f.w. of the plant.

Statistical analysis
The differences between treatments for different variables measured were tested with the two-way analysis of variance (Anova), followed by Tukey’s HSD test at significance of (p ≤ 0.05).

RESULTS AND DISCUSSION

As indicated by the numerical values, the effect of nanoparticles on germination processes varies and it depends on the type of metal. AgNPs did not inhibit the germination process as opposed to CuNPs and AuNPs. In both variants of the experiment the number of germinated seeds on the medium supplied with AgNPs was higher than on the control medium. These results suggest  that the application of silver nanoparticles has enhanced the seed germination potential [14]. While included in the medium CuNPs and AuNPs reduced the germination efficiency respectively by 31% , 24% and 5%, 19% with respect to control (Tab. 2). The experiments indicate and confirm that some nanoparticles have negative effects on seed germination. The toxicity effect of nanoparticles on seed germination after adding metal nanoparticles to the soil  was reported also by Shah and Belozerova [34]. Ma et al. [21] obtained similar results in ex vitro tests , but that indicator might not be sensitive enough to assay the toxic effects of  metal nanoparticles on plants. The best results are obtained by monitoring other growth parameters, including the shoots or roots regrowth rate [15, 19]. Roots have a direct contact with metal nanoparticles. The nanoparticles should have an essential impact on root development and growth. Our study shows that nanoparticles at this concentrations do not affect the roots length. However, there was an increase in the root length due to nanosilver and nanocopper on unsterilized medium. The roots were about 0.25 and 2.0 cm longer then on the control medium, and in the case of gold on unsterilized medium, the length of the root decreased by 1,75 cm, although the difference was non-significant. Similar trends were also found for the root length by Hojjat and Hojjat [14] and Sharma et al. [35]. In studies conducted by Sharma and coworkers [35] seeds of Brassica juncea, were germinated on MS medium supplemented with silver nanoparticles at different concentration. Seven-day-old seedlings were used for the experiments. Autors reports about increase in shoot and root length of Brassica juncea seedlings treated with high silver nanoparticles concentration. The root length of Trigonella foenum in ex vitro conditionswas higher on the medium with 10 μg mL^-1AgNPs as compared with no AgNPs control, however the roots growth decreased with increasing concentrations of AgNPs [14]. Additionally, as for the root morphology, plants roots were long and showed many rootlets on the control medium, whereas the plants exposed to AgNPs demonstrated  much shorter roots and fewer rootlets, as  compared to the control, and the root exposed to AgNPs was abnormally developed compared to the controls [18]. Numerous studies show that as the higher the silver nanoparticles concentration, the significantly lower the root length [1, 19, 44]. Similar observations have also been reported for TiO₂ nanoparticle-treated spinach seedlings [46]. Significant differences in rapeseed growth were reported in any cases when metal nanoparticles were used. As for the sterilized medium, AgNPs decreased the rapeseed height even by 3.82 cm as compared with the control, the slightest growth limitation  was recorded for AgNPs+CuNPs (by 1.87). The inhibitory effect was slightly stronger for  the unsterilized medium. AgNPs decreased the average plant height by 5.87 cm, the lowest growth has reduced CuNPs by 2.67 cm in comparison to the control. The use of AuNPs in both variants of the medium also had an inhibitory effect on the height of the seedlings and was respectively 2,87 cm and 3,8 cm. Sharma et al. [35] report on a decrease in the shoot length being affected by a high silver nanoparticles concentration; 100 mg kg^-1 and higher. The toxicity of AgNPs to crop plant species Phaseolus radiatus and Sorghum bicolor in agar and soil mediumwas measured by Lee and coworkers [18]. P. radiatus and S. bicolor in agar media showed AgNPs concentration dependent-growth inhibition. Bioavailability and effect AgNPs were less in soil than agar. It was observed that the growths of P. radiatus and S. bicolor were adversely effected by increasing the AgNPs exposure concentrations.

However, in the seedlings of Arabidopsis thaliana on MS medium, both inhibitory [36] and stimulatory silver nanoparticles effects [32] were observed. There is a suggestion that, although inducing a free radicals accumulation, nanosilver can enhance the root growth [45]. It is possible that nanoparticles sensitivity dependends on plant species.

Considering the phytotoxic effect of nanoparticles, the Authors have studied the influence of nanoparticles on different physiological parameters; the total chlorophyll and carotenoid content and the activity of antioxidant enzyme. Total chlorophyll and carotenoid content decreased after the exposure to metal nanoparticles. This effect was particularly noticeable after the application of AuNPs in the unsterilized medium. The exposure to AuNPs resulted in a 33% reduction in the chlorophyll content, however the lowest decrease in the chlorophyll content was observed in nanocopper (only by about 17%). A similar reducing concentration effect for nanogold was obtained for carotenoids in plants derived from the sterilized medium. The amount of carotenoids after applying that factor decreased by 28% (Tab. 3). Although Arora et al. [4] observed that the gold-nanoparticle treatment increased the total chlorophyll content in the seedlings of Brassica juncea under field conditions and accounted for the process by referring to limiting the inhibitory effect of ethylene on the chlorophyll content. The high chlorophyll content was associated, probably, with another nanoparticles application method, as compared to our experiment. The total chlorophyll and carotenoid content in the present study was reduced due to AgNPs. The effect of AgNPs on the physiological response of the plant was covered by numerous studies. Others [12, 13, 31] observed a decrease in the total chlorophyll and carotenoid content in the plant of Pelargonium graveolens and Solanum lycopersicum exposud to the high nanosilver concentration (80 mg·L^-1).

A good marker of oxidative stress is not only the chlorophyll content itself [5, 10] b ut also the ratio of chlorophyll a to chlorophyll b (chl a/ chl b) [5, 41]. In our experiment the ratio of chl a / chl b in rapeseed plants decreased significantly as compared to the control plants as a result of the use of nanoparticles. In all the variants it was due to decrease in the content of chlorophyll a (Tab. 3). Decrease in the ratio of chl a/ chl b, which is the effect of a lowered content of chlorophyll a, can lead to serious damage to photosystems and related photoinhibition [7]. The reduction in the photosynthetic pigments content may be attributed to the toxic effect of the high NPs concentrations.

The nanoparticles treatment induced the activities of specific antioxidant enzymes such as catalase, resulting in reduced reactive oxygen species levels [35]. The results of research by Nenghui et al. [26] show the catalase (CAT) activity being suppressed by Cu treatment, which leads to oxidative damage and inhibits seed germination. Silver nanoparticle clusters show an efficient catalytic activity in redox reactions by acting as electron relay centers, behaving alternatively as an acceptor and donor of electrons. An effective transfer of electrons is facilitated when the cluster redox potential is intermediate between the electron donor and the electron acceptor system [22, 35]. Gold, silver and copper nanoparticles indicated formations of ROS, resulting in an increased catalase activity. The highest catalase activity was observed in the variant with AuNPs (8.167 μmol H₂O₂ min^-1 g^-1 f. w.) in the unsterilized medium (Fig. 1). AuNPs increased the activity of catalase as compared with the control. High temperature and pressure which occur during media sterilization reduced the biological activity of nanoparticles. The reduction in catalase activity in sterilized medium as compared to the unsterilized medium depends on nanoparticles and it was 47% for AgNPs, 58% for AgNPs+CuNPs, 71% for Cu and 32% for AuNPs. Thus, the nanoparticle effect depends on the type of nanoparticles [29, 35, 45]. The lowest catalase activity was observed in the variant with AgNPs+CuNPs (1.667 μmol H₂O₂ min^-1 g^-1 f. w.) in both variants of the experiment. Apodaca et al. [3] observed a reduced catalase activity in the leaves of kidney bean plants cultivated in soil treated with 100 mg·kg^-1 of CuNPs. Also, Taran et al. [37] observed a catalase activity decrease in all the variants of the colloidal solution of metal (Ag, Cu, Fe, Zn, Mn) nanoparticles used to define the adaptive responses in plants. All that suggests a need for further research to understand the metabolic response of plants following the exposure to different combination of nanoparticles.

CONCLUSION

To conclude, these results reveal that the application of silver nanoparticles can enhance the seed germination potential. The application of nanoparticles did not affect the root length significantly, however it decreased the shoot growth. It was found that the size of the individual growth parameters depended on the exposure to certain types of nanoparticles. The total chlorophyll, the ratio of chl a/ chl b and carotenoid content decreased after the exposure to metal nanoparticles. Oxidative stress in the nanoparticle-treated plants was reflected by an increased catalase activity in rapeseed seedlings. Gold nanoparticles increased the activity of catalase as compared with the control. Analyzing the numerical results suggest a conclusion that the high temperature and pressure occuring during media sterilization affect the reduction of the biological activity of nanoparticles, therefore a lower catalase activity was recorded in plants from the sterilized medium. It is particularly evident for the ratio of chl a/ chl b and catalase activities. Despite a growing number of research on the biological effects of nanoparticles on land plants, we should continue to explore these areas of knowledge. The application of nanoparticles in in vitro cultures can be very important to understand the nanoparticles toxicity for plants.

REFERENCES

Accepted for print: 20.12.2018

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