1. INTRODUCTION
Numerous industries, including those in pharmacology, farming, food technology, and environmental protection, use nano-particles [1]. Due to their unique physical-chemical features, Nanoparticles (NPs) are gaining more attention in agricultural applications to augment plant development and yield. Nanomaterials easily enter different plant tissues and cells due to their small size, allowing for targeted delivery and precise release [2]. Despite these benefits, more studies have shown the inhibitory effects of nanomaterials on plants, including cellular membrane disruption, the release of heavy metal ions, the generation of reactive oxygen species (ROS), and alterations to DNA and proteins [3]. Different kinds of man-made nano-particles have been examined for their valued effects on the progress, productivity, and quality of important crops as nano-pesticides, nano-sensors, and nano-fertilizers [4]. To boost seedling growth and stress tolerance, which, in turn, increases plant productivity and food’s nutritional value, NTs are also being used as a coating agent, an exercise known as “nano-priming,” for seeds [5-7]. These nano-priming advantages are related to enhancements in seed germination, as well as changes in several biochemical and physiological and signaling pathways in seeds, like upholding ROS homeostasis [6,8-11]. Inquiries of nano-priming effects on seed germination have made increased use of NPs worldwide, such as zinc oxide and zinc, silver, iron, and titanium oxide nano-particle [12-16]. Furthermore, under drought stress conditions, nano-priming with Multi-walled carbon nanotubes (MWCNTs) improved the percentage of seed sprouting for Dodonaea viscose and Alnus subcordata seeds [17,18].
CNTs, for example, have been shown in studies to penetrate plant roots and then translocate to the vascular bundles and aerial part through the xylem through the transpiration process [19]. They can also penetrate the cell wall and membrane through pores or channels formed by the apoplastic route and endocytosis, allowing them to act as carriers of substances or chemicals within cells [20].
Pennisetum glaucum (L.) R.Br. (Pearl millet), commonly referred to as bajra, is a grain crop that is grown generally in tropical, dry regions of Asia and Africa. It is a large genus of the Poaceae family, with about 140 species that thrive in a variety of environments worldwide [21]. It is regarded as one of the most important farming crops, with a 6th rank worldwide [22]. The most basic food source for families in the world’s undeveloped nations and among the unfortunate people is pearl millet. Among millets and grains, it is also one of the crops that can endure drought the best. More than 90 million people eat pearl millet, which is grown on 27 million hectares around the world [23]. It can be full-grown in drastic conditions; meanwhile, it is well adapted to production systems with low soil fertility, high temperatures, and low rainfall (200–600 mm) [24]. Millet grains contain more micronutrients such as iron and zinc than maize, rice, wheat, and sorghum, as well as more fiber (1.2 g/100 g), a small amount of starch, and 8–19% protein [25]. To make bread, pasture, flour, and couscous, pearl millet is utilized [26]. It is majorly cultivated to produce silage, green chop, pasture, hay, and stand over fodder that is directly eaten as cattle fodder [21,27].
Pearl millet, a climate-resilient crop with a high nutritional value, can be used to improve nutritional quality and combat malnutrition. It is almost free of major diseases and insect attacks and could be grown with a good yield [28].
Fullerenes, graphene, and nanotubes (NTs) are some of the carbon nano-materials among other nano-particles [29]. According to reports, CNTs promote seed germination [30-32]. Contrary effects on seed germination result from the fact that the effect of CNTs depends on conc., the solubility of applied nano-material, and size [30]. Moreover, it has been shown that CNTs can lessen the adverse effect of different abiotic stressors together with salinity revealing their efficiency to augment productivity and quality under unfavorable conditions [31].
Martínez-Ballesta et al., 2016, demonstrated that broccoli plants (Brassica oleracea) treated with MWCNTs significantly increased their growth (32.7%), and a positive effect was also observed under saline stress conditions [33]. MWCNTs improved water absorption in seeds through aquaporins and cation exchange in the cell wall matrix, increasing nutrient concentrations such as Ca and Fe. These minerals can help with germination and plant growth and development, which can have an impact on performance [34]. Furthermore, the interaction of CNTs with proteins and polysaccharides was discovered to cause a cascade of signaling that resulted in the accumulation of compounds that led to cell wall thickening and subsequent growth [35].
It is especially important to look for new nanotechnology-based alternatives that allow for improvements in agricultural crop operation and are simple to implement. As a result, it is critical to investigate the impact that carbon nanomaterial can have on production systems in order to improve them. In the present study, seedling growth, proline content, lipid peroxidation, chlorophyll content, indole acetic acid (IAA) content, and seed germination level of P. glaucum seedlings treated with diverse concentrations of CNTs were studied. The use of MWCNTs for the growth of pearl millet can be better understood by the results obtained in this study.
2. MATERIALS AND METHODS
2.1. Plant Material
Seeds of P. glaucum (L.) R.Br. were procured from the Rajasthan Agricultural Research Institute (RARI) Durgapura, Jaipur, Rajasthan with a variety of Rajasthan hybrid (RHB) 173.
2.2. Dispersion of Nanomaterial and Seed Treatment
MWCNT Type 10 Carbon NTs (CNTs) (-COOH Functionalized) has been procured directly from the company named sisco research limited (SRL Pvt. Ltd), India. The Specifications for MWCNTs are approximately 95% pure, 10–20 nm in diameter, and 10–30 μm long and suspended using ultrasonication as defined earlier in Park et al., 2014 [32]. Without combining any surfactants, 200 mL of autoclaved distilled water was mixed with 200 mg of nanomaterials before being subjected to ultrasonic vibration at 150 W and 40 kHz for 30 min. in a sonicator. Before use, a stock of MWCNTs was diluted with many concentrations of autoclaved distilled water.
The pearl millet seeds were carefully cleaned with 20% [v/v] Extran® (Merck, India) for 3 to 4 min. followed by washing with distilled water 3 times. The seeds were surface disinfected with a Laminar air flow hood fitted with UV light using a 0.1% HgCl2 [Merck, India] solution for at least 3 min, and then washed 3 times with autoclaved distilled water. Further, different concentrations of multi-walled CNTs (MWCNTs), including 30 ppm (MW30), 60 ppm (MW60), 90 ppm (MW90), 120 ppm (MW120), and 150 ppm (MW150) were prepared from the stock solution for the experiment. Seeds were imbibed in the prepared nano-suspension for 24 h at 100 rpm in a shaking incubator. The experiment was done in triplicate and the seed incubated in pure de-ionized water was used as a control, denoted by C0.
2.3. Seed Germination and Seedling Development
The blotting paper method was used to identify seed viability tests. Treated pearl millet seeds were placed (10 seeds per Petri plate) in glass Petri plates having germination beds saturated with distilled water. To prepare the germination bed, an autoclaved absorbent cotton sheet of 3–5 mm in thickness was placed on the Petri plate, which was then covered with autoclaved filter paper cut into the same shape, and 5 ml of autoclaved distilled water was added, following the instructions of the International Seed Testing Association [36]. This whole exercise was done under laminar airflow to avoid the unwanted growth of microorganisms on the germination bed as well as germinating seeds, which can alter the results significantly. The Petri plates were kept in a growth compartment with a temperature control of 26 ± 1° C, a controlled dark/light cycle of 16/8 h, and the illumination of 24 mol/m2/s for 15 days. After that, measurements of morphological factors such as shoot and root length, fresh and dry weight, and seed germination were made.
2.4. Proline Content
The Bates et al., 1973, method was used to assess proline. The 0.5 g expunged seedling was crushed in 5000 μL of 3% aqueous sulfosalicylic acid using mortar and pestle after being refrigerated in a deep freezer at −20°C [37]. The even mixture was then centrifuged for 10 min at 13,000 rpm at a temp. of 4°C. A reaction mixture was formed by mixing 2000 μL of supernatant with 2000 μL each of acid ninhydrin and glacial acetic acid. At 100° C, this reaction mixture was heated for 1 h. By keeping it in the ice bath for 5 min. after this treatment, the reaction was completed. After that, 4000 μL of toluene was added, and a UV-visible absorbance was taken at 520 nm with a spectrophotometer (UV-1800, Shimadzu). 1 mg/mL of proline was used as the standard in glacial acetic acid. By using a standard curve, the unknown concentration of the proline was estimated as mmole/g F.wt.
2.5. Lipid Peroxidation
Calculating the gathered malondialdehyde (MDA) by the procedure described by Heath and Packer., 1968 followed for the study of lipid peroxidation [38]. After homogenizing 0.2 g of pearl millet seedlings in 2000 μL of 5% TCA, the sample was centrifuged for 15 min. at 10,000 rpm. Following that, 1 mL of 0.5% 2-Thiobarbituric acids was added to 1 mL of supernatant (prepared in 20% TCA). This solution was heated in a boiling water bath for 30 min. After that, it was rapidly cooled on ice to stop the reaction and then centrifuged for 10 min. at 10,000 rpm. By taking the absorbance at 600 nm, the absorbance at 532 nm was attuned for non-specific turbidity. An extinction coefficient of 155 mM-1 cm-1 was used to determine the quantity of MDA. The values were given in terms of mM MDA/gm F. wt.
2.6. Total Chlorophyll Content
To determine the total chlorophyll content, the Arnon method., 1949 was used [39]. 0.5 g of fresh leaves of seedlings were collected and after that, chilled in a deep freezer (−20°C). By homogenizing the leaves in 80% acetone and centrifuging them for 10 min at 10,000 rpm, the pigments were extracted. The supernatant was collected for pigment estimation. The pigment analysis was performed by spectrophotometer immediately after the supernatant was separated. At 663 and 646 nanometers, absorbance was measured. As a blank, acetone at 80 % was used. To estimate the total chlorophyll conc., Lichtenthaler and Wellburn’s [40] equations method was used: Chlorophyll a = 2.81A646−12.21A663, Chlorophyll b = 5.03A663−20.13A646, Total chlorophyll content = Chlorophyll a + Chlorophyll b, where, A646 and A663 are the absorbance at 646 nm and 663 nm wavelengths, respectively. The results were expressed as mg per gm F.wt.
2.7. IAA Content
The conc. of IAA was assessed using the Gordon and Weber technique [40]. After being chilled in a deep freezer, 1 g of seedlings was crushed in 10 mL of PO4 buffer (0.05M) of pH 7.0. This homogenate was centrifuged for 15 min. at 10,000 rpm and 4°C. Then, after centrifuging once more at 5000 rpm for 10 min, 30% cold acetone was added to 70% of the supernatant to a final concentration of 100%. After mixing 1000 μL of supernatant with 2000 μL of Salkowaski reagent, the mixture’s optical density (OD) at 530 nm was measured after 25 min. To determine the unidentified IAA concentrations, the OD value was plotted on the standard curve. IAA (1 mg/mL) was used to make the standard curve. The IAA content was specified in terms of μg/g.
3. RESULTS AND DISCUSSION
3.1. Seed Germination
The crop production depends on fast and uniform seed sprouting [41]. When pearl millet seed was treated with MWCNTs at MW30-MW150 concentrations, higher germination was determined at MW90, that is, 80% germination. Further germination of seeds declined at maximum MW120 and MW150 CNTs levels, compared to control seeds treated with sterilized distilled water. Germination increased by raising MWCNTs concentration to MW90. Consequently, it was observed that MWCNTs work as a promoter at a certain level after they can generate stress conditions for seed germination. According to Lin and Xing., 2007, seed germination rates of Brassica napus, Raphanus sativus, Lolium perenne, Zea mays, Lactuca sativa, and Crocus sativus improved by MWCNTs treatment [42]. The improvement in germination rates of pearl millet seeds by AgNPs was also reported by Parveen and Rao., 2015 [43]. Carbon nanomaterial-treated has the potential to uptake more water than untreated seeds that are responsible for increasing germination rates [44]. However, in the study by Park and Ahn., 2016, the germination of carrot seed treated with MWCNTs showed a 25% reduction concerning control [41]. This can occur because of the blocking of seed coat pores with micron-sized sheets of graphene that lead to cause less water permeability and a reduction in germination [44]. According to Thuesombat et al., 2014, sprouting and germination rate of rice are found to be affected by the level of dose of nanoparticles (NPs) [45]. Thus, the results of this study suggested that improvement in seed germination by MWCNTs treatment is exhibited in a dose-depended manner.
3.2. Seedling Growth
Plants can quickly respond to NTs since roots are the chief tissues through which NMs enter plants [41]. The root length of pearl millet was increased up to (15.3 cm) at MW90 treatment of MWCNTs. At MW120 and MW150 length of pearl millet roots drastically decreased to 6.1 cm and 4 cm, respectively, in comparison with the control, which have a 13 cm root length. Likewise, among all concentrations of MWCNTs treatment, MW90 led to the highest shoot length at 2.7 cm, followed by MW60 with 2.5 cm and MW30 with 1.7 cm as compared to the shoot length of untreated seeds (1.6 cm) in Figure 1.
 | Figure 1: Effect of Multi-walled carbon nanotubes on shoot length and root length development of Pennisetum glaucum at different concentrations at 15 days of the seedling stage. [Click here to view] |
Above the MW90, the length of the shoot was found to be increased as specified in Figure 1. Likewise, Shekhawat et al., 2021, noted a little rise in the shoot and root length of Vigna radiata with 5.67% and 16.65%, respectively, at 100 μM level of CNTs, compared to control, but at higher concentrations, both lengths declined [46]. At all provided MWCNTs levels, length of shoot-root and seed vigor index of Dodonaea viscosa under drought stress conditions were reported with the highest values by Yousefi et al., 2017, in comparison with control [18].
Rahimi et al., 2016, also revealed positive results of the shoot and root length, d.wt., and seed vigour index of A. subcordata seedlings under drought conditions at 0–100 mg/L content of MWCNTs [17]. According to Li et al., 2016, the length of the root and shoot was increased up to 0.4 mg/mL of fluorescent C dots before being reduced at higher treatment levels [47]. In a study by Nair et al., 2012, rice seeds treated with 50 g/mL CNTs had the lengthiest roots and shoots compared to the control group [44]. However, Canas et al., 2008, reported that CNTs treatment enhanced the root length of Allium cepa and Crocus sativus but reduced the elongation of lettuce and tomato roots [48]. It was observed due to the differential response of nanoparticles on plant species or genotype. In addition, the impact of MWCNTs treatment on the fresh weight of pearl millet seedlings was higher with 0.09 g at MW90. A slightly increment with 0.06 g was observed at MW30 compared to the control (0.05 g). Drastically reduction in fresh weight was recorded comparatively at MW120 and MW150 treatment with 0.01 gm and 0.001 gm, respectively, Table 1.
Table 1: Morphological parameters of Pennisetum glaucum 15 days seedlings treated with variable levels of multi walled carbon-nanotubes.
| Sample | Shoot length (cm) | Root length (cm) | Fresh weight (gm) | Dry weight (gm) |
|---|---|---|---|---|
| C0 | 1.6±0.12b | 13±0.09d | 0.05±0.01b | 0.007±0.001c |
| MW30 | 1.7±0.16b | 13.4±0.18c | 0.06±0.01b | 0.008±0.0004bc |
| MW60 | 2.5±0.21a | 14.1±0.02b | 0.07±0.02ab | 0.0089±0.0002ab |
| MW90 | 2.7±0.10a | 15.3±0.21a | 0.09±0.01a | 0.0096±0.0001ab |
| MW120 | 2±0.24b | 6.1±0.14e | 0.01±0.01c | 0.006±0.0006d |
| MW150 | 0.5±0.10c | 4±0.29f | 0.001±0.003c | 0.0003±0.0002e |
*All the triplicate values are present in Mean±SD; F.wt; Fresh Weight. Means with different superscripts within same parameters based on different concentrations are significantly different (P < 0.05).
Similarly, in Figure 2, the maximum dry weight of MWCNTs treated P. glaucum was observed at MW90 with 0.0096 g and thereafter declined up to 0.0003 g at MW150. The average weight of dry seedlings was recognized with 0.008 g and 0.0089 g, at MW30 and MW60, respectively.
 | Figure 2: Morphological parameters of Pennisetum glaucum 15 days seedlings treated with variable levels of multi walled carbon-nanitubes (MWCNTs). [Click here to view] |
Shekhawat et al., 2021, reported 1.20 and 1.14 times higher fresh and dry weight of V. radiata at 100 μl MWCNTs content than the control [46]. The fresh weight of Brassica juncea seedlings was augmented at 50 mg/l AgNPs [49]. Applications of CNTs, according to Liang et al., 2013, enhance the concentration of potassium and nitrogen in plant organs, which is responsible to plant growth improvement [50]. A reduction in plant growth at higher CNTs levels can be because of CNTs accumulation on the roots that control the permeability of plant minerals [51].
3.3. Proline Content
Common physical responses in plant cells uncovered to variable environmental conditions comprise the gathering of proline [46]. The improved concentration of proline in pearl millet treated with variable levels of MWCNTs is described in Table 2.
Table 2: Bio-chemical analysis of Pennisetum glaucum 15 days old seedlings treated with variable levels of carbon nanotubes.
| Sample | Proline content (µmole/g F.wt.) | LPO assay (µM MDA/g F.wt.) | Total chlorophyll content (mg/g F.wt.) | IAA content (µg/gm) |
|---|---|---|---|---|
| C0 | 0.32±0.01f | 0.29±0.02c | 14.88±0.27f | 21.5±0.37f |
| MW30 | 0.46±0.005e | 0.26±0.01d | 20.8±0.21e | 25.12±0.10e |
| MW60 | 0.59±0.01d | 0.21±0.02e | 26.48±0.05b | 28.12±0.03d |
| MW90 | 0.67±0.02c | 0.15±0.005f | 27.74±0.15a | 33.37±0.02c |
| MW120 | 0.72±0.005b | 0.45±0.01b | 23.023±0.08c | 34.75±0.082b |
| MW150 | 0.84±0.02a | 0.59±0.005a | 21.24±0.01d | 37±0.05a |
*All the triplicate values are present in Mean±SD; F.wt; Fresh Weight. Means with different superscripts within same parameters based on different concentrations are significantly different (P < 0.05).
The proline content was significantly augmented by the gradually raised carbon- NTs concentration. As in Figure 3, the maximum level of MWCNTs (MW150) showed a 0.84 μmole/gm F.wt. concentration of proline, which is the highest as compared to other treatments. Control showed 0.32μmole/gm F.wt. proline content. When exposed to 100 μM CNTs, Shekhawat et al., 2021 found that the proline levels in V. radiata leaves and roots were 1.48 and 6.29 times, those in the control group [46]. The enhancement of proline concentration with increasing CNTs treatment indicates water stress conditions due to variations in cell permeability [52,53]. As scavengers of ROS, plants often endorse a variety of total antioxidant activity, such as thiols, carotenoids, and proline, as well as enzymatic antioxidant activity, like catalase, superoxide dismutase and peroxidase [54-56].
 | Figure 3: Bio-chemical analysis of Pennisetum glaucum 15 days old seedlings treated with variable levels of multi walled carbon-nanotubes (MWCNTs). [Click here to view] |
3.4. Lipid Peroxidation
MDA, a byproduct of LPO, serves as an oxidative stress indicator. Consequently, under stress conditions, the MDA level is augmented in the plant [57]. With an exact dosage of MWCNTs up to MW90, the enhancement of MDA content was seen in the recent learning. As in Figure 3, the gradual reduction in MDA concentration was determined at MW30, MW60, and MW90 with 0.26, 0.21, and 0.15 μM MDA/g F.wt. as compared to control (0.29 μM MDA/g F.wt.). A progressive increment in MDA level was recorded at MW120 and MW150, that is, 0.45 and 0.59 μM MDA/g F.wt. as specified in Table 2. The concentration of MDA in both the leaves and the roots of V. radiate was reduced in Shekhawat et al., 2021, study up to 100 Molar of CNTs and further at 150 Molar [46]. With leaf and root sections, MDA was 1.09 and 1.06 times higher. Due to the small size of carbon nanoparticles, which have better diffusion capacity in plant cells and enhance nutrient availability, LPO can be reduced to a certain amount of CNTs. Nutrient absorption augments the tolerance ability of plants under stress conditions; therefore, the MDA level is reduced [58]. But at higher concentrations of CNTs, MDA content increases due to the stimulation of CNTs-mediated oxidative stress [59].
3.5. Total Chlorophyll Content
Total chlorophyll conc. has a vital role in the determination of plant development and commonly it is a sign of detrimental effect on the plants [51]. According to El-Badri et al., 2021, nano-priming reduces biosynthesis pigments and ROS by alleviation of O2− and H2O2 accumulation and chlorophyll degradation along with enhancement of anti-oxidant to increase the defensive mechanism in the plant [16]. The impact of CNTs on the chlorophyll level of pearl millet leaf was evaluated in this study. As compared to the control (14.88 mg/g F.wt.), the maximum concentration of chlorophyll, that is, 27.74 mg/g F.wt., was recognized at MW90. At MW150, chlorophyll level declined significantly up to 21.24 mg/g F.wt. comparatively as mentioned Table 2. Consequently, it was suggested that MWCNTs facilitate the synthesis of chlorophyll in pearl millet leaves at a particular concentration, and higher levels of MWCNTs lead to cause a negative impact on chlorophyll content. Likewise, Shekhawat et al., 2021, lead a study in which a steady rise in total chlorophyll concentration was observed up to 100 μM of CNTs treatment, followed by a reduction recognized at higher 150 and 200 μM concentrations of carbon-nanoparticles [46]. The application of MWCNTs (50 mL) in carrot leaf with 25–30% enhancement in chlorophyll level was recorded by Park and Ahn, 2016 [41]. In the research, it has been also reported a 14.8% increment in chlorophyll biosynthesis in mung bean sprouts by carbon dots. This is most likely because CNTs improve the activity of the photosystem (Rubisco activity and chlorophyll concentration) by increasing the rate of e-transfer. But at higher CNTs levels, reduction in chlorophyll synthesis occurs due to the direct association of engineered nanoparticles with the chloroplast and oxidative stress takes place in the cell’s plastid [51].
3.6. IAA Content
For plant development and growth, IAA is an essential plant hormone [60]. Many auxin-responsive genes are controlled at the transcriptional level in plants with the help of auxin through auxin/indole-3-acetic acid-Auxin Response Factor (ARF) auxin-signaling modules [61]. Therefore, proper IAA production, signaling, and transport are vital for rooting [62]. As shown in Table 2, when the exposure level of MWCNTs was amplified from MW30 (30ppm) to MW150 (150ppm) in comparison to control, the amount of IAA in pearl millet seedlings progressively improved. The control showed 21.5μg/gm IAA that was raised to 37μg/gm at MW150 higher quantity of MWCNTs. Li et al. [60] reported 39% and 56% higher endogenous IAA concentrations in shoots and root parts of wheat treated with chitosan nanoparticles. Cheng et al., 2016, detected an increment in the abundance of ARF and IAA in B. napus by 50 mg/l of graphene oxide [63]. Sun et al., 2020 reported enhancement of auxin contents, stem cell niche activity, signaling of root apex, and potential of meristematic cell division of Arabidopsis by treatment with nano priming application of SWCNTs [64]. Thus, the result of this study indicated that MWCNTs treatment can increase IAA content in pearl millet at a particular concentration.
4. CONCLUSION
The growth and biochemical parameters of pearl millet seedlings improved by the use of MWCNTs. This study shows that MWCNTs affect plant development differentially. At a specific concentration of MWCNTs, 90 ppm (MW90), the height of the shoot and root as well as the fresh weight and dry weight of pearl millet seedlings were augmented. Above the MW90, the length and biomass both declined. Likewise, a significant improvement in LPO, proline, chlorophyll, and IAA content was also identified at MW90. However, at higher concentrations of MWCNTs, proline, and IAA concentrations were increased; it could be possible due to, regulating the stress condition. In the present work, it was observed that a certain MWCNTs level (MW90) favors the growth of pearl millet seedlings.
Seed priming with MWCNTs elicited favorable responses, which may improve the development of the pearl millet crop. These findings suggested that treating pearl millet seeds with MWCNTs could be a good way to induce biostimulation while also demonstrating an easy method of application. Overall, the impact of CNTs on the development of pearl millet seedlings was strongly dependent on application rates.
5. ACKNOWLEDGMENT
We extend our appreciation towards Amity University Rajasthan, Jaipur, India, for its valuable support throughout the work.
6. AUTHORS’ CONTRIBUTIONS
All authors made substantial contributions to conception and design, acquisition of data, or analysis and interpretation of data; took part in drafting the article or revising it critically for important intellectual content; agreed to submit to the current journal; gave final approval of the version to be published; and agreed to be accountable for all aspects of the work. All the authors are eligible to be an author as per the International Committee of Medical Journal Editors (ICMJE) requirements/guidelines.
7. FUNDING
The study was financially supported by the Department of Science and Technology, Govt. of India, New Delhi for providing FIST grant (SR/FST/LS-l/2017/56) to Amity Institute of Biotechnology, Amity University Rajasthan, Jaipur, India.
8. CONFLICTS OF INTEREST
The authors declare that there are no conflicts of interest.
9. ETHICAL APPROVALS
This study does not involve experiments on animals or human subjects.
10. DATA AVAILABILITY
All the data is available with the authors and shall be provided upon request.
11. PUBLISHER’S NOTE
This journal remains neutral with regard to jurisdictional claims in published institutional affiliation.
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