Promotion of seed germination and seedling growth of Zea mays by magnesium hydroxide nanoparticles synthesized by the filtrate from Aspergillus niger

2.1 Materials

Zea mays (maize var. NMH-3493) purchased from Nirmal seeds Pvt. Ltd., Jalgaon, Maharashtra State, India were used for the study. Other chemicals such as magnesium sulfate (MgSO₄), sodium hydroxide (NaOH), Murashige and Skoog (MS) medium, etc. were purchased from Hi-media Pvt. Ltd., Mumbai and used without further purification.

2.2 Synthesis and characterization of Mg(OH)₂NPs

Modified co-precipitation method was used for the synthesis of Mg(OH)₂NPs (Singh et al., 2011). 0.1 M aqueous solution of MgSO₄ was challenged with cell free filtrate of Aspergillus niger on magnetic stirrer with continuous stirring followed by addition of NaOH (1 N) till the pH of the solution reached 8. The stirring was continued for half an hour. The white precipitate obtained by centrifugation at 4000 rpm for 30 min was collected and dried in hot air oven to get powder. The resulting Mg(OH)₂NPs powder was used for further analysis.

Further, the Mg(OH)₂NPs were characterized by Nanoparticles tracking analysis (NTA), Zeta potential analysis, X-ray diffraction Crystallography (XRD), Fourier Transform Infra- Red spectroscopy (FTIR) and Transmission Electron Microscopy (TEM).

2.3 Effect of synthesized nanoparticles on germination of Z. mays seeds

Under aseptic condition, seeds of Z. mays were surface sterilized with 0.1% mercuric chloride for 10 min followed by three times washing with sterile distilled water. Five sterilized seeds were inoculated on sterile cotton bed in each sterile petri plates (in triplicate per treatment mentioned below). The cotton bed was prepared by placing Whatman filter paper No. 1 on the sterile cotton and moisture was maintained by adding sterile distilled water. The number of germinated seeds [seeds showing emerging radicle (Luo et al., 2018)] was studied daily for up to 7 d. At the end of this experiment, shoot height and root length were measured. The effect of different concentrations of Mg(OH)₂NPs viz. 50, 100, 200, 300, 400, 500, 600, 700, 800, 900 and 1000 ppm was evaluated on growth of Z. mays and compared with control (untreated). Various parameters of germination such as Germination percentage (GP), Germination rate index (GRI), and Mean germination time (MGT) were also studied (de Souza et al., 2013).

From the germination counts, germination indexes were calculated to analyze germination percentage (GP), germination rate index (GRI) and mean germination time (MGT) after 7 d as follows:

• Germination Percentage (GP) = (germinated seeds /total no of seeds) × 100

• Mean Germination Time (MGT) = Ʃ number of germinated seeds until the 7th day × number of days from sowing germination until the day of measurement (from the start of experiment)/total no of seeds

• Germination Rate Index (GRI) = No. of seed germinated on 7th day/day of germination

2.4 In vitro efficacy of Mg(OH)₂NPs on Z. mays seedlings

The experiments were performed in two ways. In first experiment, seeds were germinated on half Murashige and Skoog’s (MS) medium. Seven day old seeds with emerging radicle (seedlings) were inoculated on MS medium supplemented with two different concentrations of Mg(OH)₂NPs (200 ppm and 500 ppm). In addition, separate control (without nanoparticles) was also maintained. The inoculated seedlings were further incubated for 7 d at 28 ± 2 °C under light condition. In second experiment, seeds of Z. mays were initially treated with Mg(OH)₂NPs (200 and 500 ppm), and control (seeds without nanoparticles) were inoculated on full MS medium. The inoculated seedlings were further incubated for 7 d at 28 ± 2 °C under light condition.

2.5 In vivo effect of Mg(OH)₂NPs on growth of Z. mays seedlings

The surface sterilized five seeds of Z. mays were sown in the pot containing sterilized soil: sand in the ratio of 3:1. To study the efficacy of these nanoparticles, various sets of plants were maintained in triplicate for each treatment of nanoparticles in ppm (100, 200, 500, 1000). The treated plants were watered every day and kept in sunlight. First treatment of above mentioned concentration of Mg(OH)₂NPs and their bulk salts was given to 15 d grown seedlings and further treatments were given at the interval of 4 days (1 ml of each concentration of nanoparticles and bulk salt was given to every plant near root). Further, the chlorophyll a (chl a) fluorescence was measured with the help of Handy-PEA (Plant Efficiency Analyzer) (Hansatech Instrument Pvt. Ltd, UK) at the interval of 3 days. The heights of the plants were also measured at every 3rd d. In addition, all these plants were supplied with Hewitt’s solution (Rathod et al., 2011) after 10 and 20 d of growth. The experiment was performed for 30 d and after that root length, fresh weight (mean of weight of 5 plants) and dry weight (mean of weight of 5 plants) were measured. However, in this study, the data recorded at 30th d of experiment was considered for analysis.

2.6 Detection of nanoparticles uptake by Z. mays using atomic absorption spectrophotometer (AAS) analysis

Atomic absorption spectrophotometry was used to quantify the concentration of Mg in leaves and roots of plants. One g of dried plant sample (leaves and roots of seedlings grown on MS medium supplemented with various concentration of Mg(OH)₂NPs and plants grown in vivo were treated with 10 ml concentrated nitric acid and allowed to stand overnight. Later, solution was heated till red fumes were ceased. The solution was allowed to cool for 2–3 h, followed by addition of small amount (2 ml) of 70% perchloric acid. The solution was then transferred to volumetric flask and the volume was made up to 50 ml with distilled water. The samples were analyzed with the help of AAS instrument (Perkin Elmer-AAnalyst 300).

2.7 Estimation of chlorophyll content in nanoparticles treated Z. mays plants

For the estimation of chlorophyll content, initially 300 mg leaves (fresh weight) were partially dehydrated by soaking them in 95% ethanol for 5 min. Further, the leaves were cut into pieces, ground in mortar and pestle, mixed with 5 ml of 80% acetone in 15 ml of centrifuge tube and the mixture was incubated in dark for 30 min. These tubes were centrifuged at 4 °C for 15 min at 3000 rpm then supernatant was kept in dark. Further, the samples were analyzed with the help of UV–Visible spectrophotometer (Shimadzu UV-1800, Japan) and absorbance was recorded at 645 nm and 663 nm. Chlorophyll content (Chlorophyll a and b) was estimated by using Arnon’s formula (Arnon, 1949) mentioned below: $$\text{Chla}\left(\text{mg}/\text{g of fresh weight}\right)=\left[12.7\times \text{A}663-2.69\times \text{A}645\right]\times \text{V}/1000\times \text{W}$$ $$\text{Chlb}\left(\text{mg}/\text{g of fresh weight}\right)=\left[22.9\times \text{A}645-4.86\times \text{A}663\right]\times \text{V}/1000\times \text{W}$$ where Chl a = Chlorophyll a; Chl b = Chlorophyll b; V = volume of extract in ml; W = fresh weight of leaves in gm; A663 = the solution absorbance at 663 nm; A645 = the solution absorption at 645 nm.

2.8 Statistical analysis

The mean and standard deviation data obtained from measurement of various treatments in three replicates were statistically analyzed using ANOVA (analysis of variance) and Tukey’s multiple comparison tests to determine the significant difference among nanoparticles treatment. P values of <0.05 and lower were considered significant. Evaluations of all graphs were made using GraphPad Prism 7.03 (GraphPad Software, Inc., La Jolla, CA, USA).

3.1 Synthesis and characterization of Mg(OH)₂NPs

The modified co-precipitation method was employed for synthesis of Mg(OH)₂NPs using cell free filtrate of A. niger. The aqueous solution of MgSO₄ was reduced by the fungal extract. The formation of white precipitate at pH 8 indicated the synthesis of Mg(OH)₂NPs. The synthesis of magnesium oxide nanoparticles (MgONPs) can be performed by chemical (Camtakan et al., 2012) and biological methods (Sharma et al., 2016; Sushma et al., 2016) but to the best of our knowledge there are only a few studies available on synthesis of Mg(OH)₂NPs (Henrist et al., 2003). Moreover, in the present study, for the first time we demonstrated synthesis of Mg(OH)₂NPs using cell free extract of A. niger.

It is well known that various biomolecules are secreted in fungal filtrate, which help in the reduction of aqueous ions and form the nanoparticles. Therefore, to understand the possibility of biomolecules responsible for the reduction and capping of nanoparticles, FTIR analysis was performed. The FTIR analysis provides information on the vibrational and rotational modes of motion of a molecule, and hence it is an important technique for identification and characterization of substances. The FTIR spectrum obtained for fungal filtrate clearly shows absorption bands at 1616 cm⁻¹, 1396 cm⁻¹, 1104 cm⁻¹ which corresponds to N—H bend, CH₃—CH₃ bonding and C—N stretch, respectively. Moreover, the FTIR spectrum reported for Mg(OH)₂NPs showed absorption bands at 2116 cm⁻¹ due to —C≡C— stretch, 1629 cm⁻¹ and 1117 cm⁻¹ due to stretching of C—N (Fig. 1I). These absorption peaks correspond to various functional groups present in biomolecules (proteins), which may help in reduction of aqueous ions to nanoparticles (Agnieszka et al., 2012).

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Fig. 1 Characterization of Mg(OH)₂NPs using (I) FTIR analysis; (II) XRD analysis; (III) NTA analysis.

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The XRD patterns were recorded to investigate the phase and purity of the synthesized Mg(OH)₂NPs (Fig. 1II). The XRD pattern recorded for Mg(OH)₂NPs showed intense peak at 2θ values of 18.65, 33.99, 38.15, 50.88, 58.68, 62.06 and 68.30, which corresponds to (0 0 1), (1 0 0), (1 0 1), (1 0 2), (1 1 0), (1 1 1) and (1 0 3) planes confirming the crystalline face-centered cubic (FCC) structure. It can be indexed as the hexagonal structure of magnesium hydroxide (JCPDS file number 7-239). The significant broadening of peak indicates that the Mg(OH)₂NPs has very small grain size (Ding et al., 2001; Agnieszka et al., 2012).

Further, for size determination of Mg(OH)₂NPs, Nano tracking analysis was performed using NanoSight LM-20 (Malvern, UK). In addition, other details like mean, mode, standard deviation, and concentration of nanoparticles can also be determined using this instrument. It is generally based on tracking the Brownian motion of each particle. NTA revealed the average size of the nanoparticles to be 47 ± 36 nm for Mg(OH)₂NPs. Fig. 1(III) shows the particle size distribution histograms for Mg(OH)₂NPs. Unlike classical light scattering techniques, nanoparticle tracking analysis allows measurement of nanoparticles in suspension on a particle-by-particle basis with real time visualization, sizing, and counting at higher resolution, that allows better understanding of aggregation than other methods, such as dynamic light scattering and differential centrifugation sedimentation (Wright, 2012).

The zeta potential for Mg(OH)₂NPs was found to be −11.7 mV (Fig. 2I). Although, the zeta potential values were within the range of −25 mV to +25 mV, in biological synthesis capping of biomolecules like proteins over the nanoparticles affect the value of zeta potential (Jeevanandam et al., 2017). Hence, these values confirm high degree of stabilization of the nanoparticles. Finally, transmission electron microscopy (TEM) was performed to determine size and shape of Mg(OH)₂NPs. The TEM micrographs confirmed the formation of polydispersed nanoparticles. The size of Mg(OH)₂NPs was found to be in range of 50–100 nm (Fig. 2II). Moreover, the SAED pattern obtained (Fig. 2III) revealed crystalline structure of nanoparticles.

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Fig. 2 Characterization of Mg(OH)₂NPs using (I) Zeta potential analysis; (II) TEM analysis and (III) SAED pattern.

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3.2 Effect of Mg(OH)₂NPs on germination of Z. mays seeds

The seed germination efficacy of Mg(OH)₂NPs at various concentrations was tested on Z. mays seeds. Various parameters related of seed germination such as germination percentage (GP), germination rate index (GRI) and mean germination time (MGT) was studied. The GRI express the percentage of germination on each day of the germination period. Higher GRI values indicate higher and rapid germination of seeds.

The observations recorded have shown that the germination rate of Z. mays seeds significantly increased after treatment with Mg(OH)₂NPs as compared to control (untreated seeds). Among the treatments of various concentrations of Mg(OH)₂NPs (viz. 50, 100, 200, 300, 400, 500, 600, 700, 800, 900 and 1000 ppm), highest GP (i.e. 100%), highest GRI (i.e. 3 seeds per day) and lowest MGT (1.2 days) were observed when the seeds were treated with 500 ppm (Table 1). However, the control (non-treated seeds) maintained without treatments of Mg(OH)₂NPs showed only 41.6% of GP. The results provide evidence of the concentration dependent influence on the seed germination. Although, so far, there is no such study performed with Mg(OH)₂NPs. In a previous study, the authors demonstrated concentration dependent effect of MgONPs on seed germination of Z. mays (Jayarambabu et al., 2016). In their study, maximum seed GP (95%) was recorded at 100 ppm, whereas, control seeds showed GP of 80%. Another study reported that the carbon nanotubes can effectively penetrate through seed coat and influence the seed germination (Khodakovskaya et al., 2009).

Apart from these parameters, it was observed that seeds treated with various concentrations of Mg(OH)₂NPs demonstrated significant increase in the shoot height and root length as compared to untreated seeds. The maximum shoot height (8.2 ± 0.3 cm) and root length (14.1 ± 0.6 cm) were recorded when treated with 500 ppm of Mg(OH)₂NPs. However, seeds treated with various other concentrations of Mg(OH)₂NPs and control (untreated seeds) showed comparatively less growth of shoots and roots (Fig. 3I and II).

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Fig. 3 Effect of Mg(OH)₂NPs on seed germination (I) shoot (II) root growth.

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The above findings strongly suggest that Mg(OH)₂NPs greatly influence all the parameters of seed germination and also showed positive effect on shoot and root development. The role of silver nanoparticles (AgNPs), mixture of silicon dioxide nanoparticles (SiO₂NPs) and titanium dioxide nanoparticles (TiO₂NPs) in germination of Z. mays and Glycine max seeds was demonstrated by researchers (Lu et al., 2002; Almutairi and Alharbi, 2015) who also proposed the possible mechanism involved in seed germination. However, at the moment we don’t have direct data to support the hypothetical mechanism for seed germination in Z. mays by Mg(OH)₂NPs. But it is well known fact that Mg is a macro-nutrient essentially required for plants growth and its ions play an important role in the activation of enzymes involved in seed germination. Therefore, it is hoped that same mechanisms proposed by Almutairi and Alharbi (2015) and Lu et al. (2002) may be applied in context of Mg(OH)₂NPs.

Possibly, it is expected that Mg(OH)₂NPs attach to the waxy layer present on the surface of Z. mays seeds and nano-size facilitates the penetration into the seeds through seed coat and exerts a beneficial effect on the process of seed germination. Generally, nanoparticles increase water absorption capacity of seeds (Morales-Díaz et al., 2017). The water absorbed by seeds solubilizes gibberellic acid (GA) present in the embryo, reaches to aleuronic cytoplasm via seed tissues and produces amylase enzyme. The latter hydrolyses the seed starch into sugars (maltose) and supplies energy to the seed cells required for germination (Biazus et al., 2005; Biazus et al., 2009). In addition, nanoparticles also help to increase nitrate reductase enzyme level, leading to enhanced ability of the seeds to absorb and utilize water and other nutrients. It promotes seed antioxidant systems (Aslani et al., 2014). Apart from this, these nanoparticles may help in reduction of the antioxidant stress by reducing H₂O₂ and superoxide radicals and increase the activities of important enzymes involved in the seed germination such as superoxide dismutase, ascorbate peroxidase, guaiacol peroxidase, catalase, etc. (Siddiqi and Husen, 2017). All these changes collectively enhance the seed germination in Z. mays. Fig. 4 shows the schematic representation of possible hypothetical mechanisms involved in germination of Z. mays seeds treated with Mg(OH)₂NPs.

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Fig. 4 Schematic representation of possible hypothetical mechanisms involved in the germination of Zea mays seeds in the presence of Mg(OH)₂NPs: (1) Nanoparticles increase water and nutrients absorption capacity of seeds (2) Rapid absorption of water by seeds, it solubilizes gibberellic acid (GA) present in the embryo and get transported to seed tissue and aleuronic cytoplasm, it actives central dogma system and produces amylase enzyme. Further, amylase enzyme hydrolyses the seed starch into sugars and it supplies energy to the seed cells required for germination (3) Nanoparticles help to increase the level of nitrate reductase in embryo which enhances the seeds' ability to absorb and utilize water and other nutrients thereby promoting seed antioxidant systems and seed germination (4) Nanoparticles help in the reduction of the antioxidant stress by reducing H₂O₂ and superoxide radicals, which enhances the activities of key enzymes involved in the seed germination.

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3.3 In vitro efficacy of Mg(OH)₂NPs on Z. mays seedlings

To study in vitro efficacy of Mg(OH)₂NPs on Z. mays seedlings, two different experiments were performed. In first experiment, seeds were germinated on half Murashige and Skoog’s (MS) medium, and then seeds with emerging radicle were inoculated on full MS medium supplemented with two different concentrations of Mg(OH)₂NPs (200 ppm and 500 ppm). In addition, separate control (without nanoparticles) was also maintained.

After incubation, there was remarkable increase in the shoot height (11.96 ± 0.4 cm) and root length (3.92 ± 0.4 cm) of seedlings in MS medium supplemented with 500 ppm Mg(OH)₂NPs as compared to 200 ppm (9.0 ± 0.2 cm and 3.52 ± 0.08 cm) and control (4.08 ± 0.1 cm and 1.2 ± 0.1 cm), respectively (Fig. 5I and II).

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Fig. 5 The in vitro efficacy of different concentrations of Mg(OH)₂NPs on growth of maize seedlings supplemented with MS medium (I) shoot height (II) root length; and on initially treated with Mg(OH)₂NPs (III) shoot height (IV) root length. [Bars represent the averages of three replicates and error bars denote SDs. Statistical significance (*P < 0.05; ^**P < 0.01; ^***P < 0.001; ^****P < 0.0001) was calculated by ANOVA].

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In second experiment, Z. mays seeds initially treated with Mg(OH)₂NPs (200 and 500 ppm) and control (untreated seeds) were inoculated on full MS medium. As expected, it was found that these results were similar to the results obtained for germination study. Interestingly, in this experiment also, higher shoot height (9.32 ± 0.4 cm), and root length (8.24 ± 0.3 cm) were recorded when the seeds were treated with 500 ppm Mg(OH)₂NPs as compared to control (untreated) and 200 ppm Mg(OH)₂NPs concentration (Fig. 5III and IV).

Even after extensive literature survey, we did not find any report on in vitro study of efficacy of Mg(OH)₂NPs. But, Savithramma et al. (2012) studied in vitro efficacy of AgNPs on seedling growth of Boswellia ovalifoliolata. They supplemented the MS medium with three different concentrations (10, 20 and 30 µg/ml) and inoculated seeds. Among the three tested concentrations, seedling showed the maximum height (10.6 ± 0.3 cm) on MS medium supplemented with 30 µg/ml AgNPs as compared to other two concentrations and control (3.0 ± 0.5 cm) (MS medium without AgNPs) seedlings. Although, based on the present study, it is difficult to comment on the exact mechanism for enhanced seedling growth. The previous studies proposed that improved growth rate of seedlings may be due to enhancement in water and nutrient uptake by the treatment of nanoparticles. Savithramma et al. (2012) proposed that AgNPs incorporated in MS medium may have generated new pores on seed coat during penetration, which helps entry of moisture and water and activate nutrients inside the seeds or nanoparticles may carry the nutrients along with them, which may lead to rapid germination and growth. In the present studies also, Mg(OH)₂NPs might have developed pores on the wall.

The results obtained for in vitro study suggests that Mg(OH)₂NPs in both the experiments i.e. when supplemented with MS medium or seed treatment with these nanoparticles followed by their inoculation on MS medium showed the enhanced morphological characteristics than that of control seedlings. The probable reason for enhanced overall growth of seedlings may include easy accessibility of nutrients (macro and micro) in nanoform by plants. Further, the translocation of nanoparticles in various plant parts support the enhanced morphology and physiology of seedlings. Due to unavailability of any in vitro study concerning efficacy of Mg(OH)₂NPs on any plant, we claim that this study has been performed for the first time.

3.4 In vivo assessment of Mg(OH)₂NPs efficacy on growth of Z. mays plants

Considering the promising efficacy of Mg(OH)₂NPs in seed germination and in vitro study, we have performed in vivo study to evaluate efficacy of these nanoparticles in plant growth promotion. For in vivo study, various experimental sets in pots were maintained depending on the treatment of Z. mays with various concentrations of Mg(OH)₂NPs their salts as control. Similarly, separate control (without treatment) was also maintained as described in methodology. Further, the morphological characteristics such as root length and shoot height were measured. In addition, Chl a fluorescence measurement and analysis (biophysical phenotyping) was also made to assess effect of nanoparticles on plant growth promotion and vitality.

The biophysical phenotyping was performed on the basis of Chl a fluorescence measurement using Handy PEA. This instrument determines the behavior and performance of photosystem II (PSII) in terms of different structural and functional parameters in photosynthesis. It is mostly studied using JIP-test, which involves analysis of the chlorophyll a fluorescence transient OJIP shown by the photosynthetic plants upon illumination after dark adaptation (Kalaji et al., 2014). The Radar graph shows average JIP test value resulting from the Chl a fluorescence measurement.

The findings reported for in vivo study evidenced that Z. mays treated with Mg(OH)₂NPs showed significant growth promotion as compared to the plants treated with their respective salts (MgSO₄) and control (untreated plants). Moreover, it was also observed that the plant growth promotion was concentration dependent. When the plants were treated with different concentrations of Mg(OH)₂NPs (100 ppm, 200 ppm, 500 ppm, 1000 ppm), 500 ppm showed the significant increase in morphological characteristics i.e. shoot height (44.2 ± 1.6 cm) and root length (29.2 ± 1.3 cm) after 30 d as compared to plants treated with various concentration (100 ppm, 200 ppm, 500 ppm, 1000 ppm) of its salts and control plants. Moreover, the plants treated with 500 ppm (bulk treatment) showed enhanced shoot height (38 ± 1.5 cm) and root length (21.6 ± 2.3 cm), while the control plants demonstrated poor growth and development (Fig. 6I and II).

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Fig. 6 The in vivo efficacy of different concentrations of Mg(OH)₂NPs and their bulk counter parts on growth of maize (I) shoot height (II) root length (III) Radar plot showing effect of various concentrations of Mg(OH)₂NPs on specific energy, phenomenological fluxes and performance of plants. [Bars represent the averages of three replicates and error bars denote SDs. Statistical significance (*P < 0.05; ^**P < 0.01; ^***P < 0.001; ^****P < 0.0001) was calculated by ANOVA].

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Further, Chl a fluorescence measurement of all these experimental plants were recorded using Handy-PEA and calculated in the form of specific energy fluxes (ABS/RC, TRo/RC ETo/RC and DIo/RC), phenomenological fluxes (RC/CSo, ABS/CSo) and PIabs which are represented in graphical form (radar plot). The maximum PIabs and lowest DIo/RC were recorded for the plants treated with 500 ppm followed by 200 ppm, 100 ppm and 1000 ppm Mg(OH)₂NPs (Fig. 6III). As far as the treatment of Mg salt is concerned, the plants treated with 1000 ppm Mg salts showed highest PIabs and lowest DIo/RC. Whereas, control (untreated) plants showed much lower PIabs and higher DIo/RC as compared to both Mg(OH)₂NPs and bulk Mg treated plants. The high-performance index and less dissipation of energy in the form of heat signify good efficiency of photosystem II. Apart from these, other specific energy fluxes such as ABS/RC, TRo/RC and ETo/RC found to be more and phenomenological fluxes (RC/CSo, ABS/CSo were found to be less in plants treated with 500 ppm Mg(OH)₂NPs as compared to other treated plants (Rathod et al., 2011) (Fig. 6 III). Similar pattern was found for 500 ppm Mg(OH)₂NPs treated plants in case of specific energy fluxes and phenomenological fluxes. Further, we have studied whether there is any correlation between morphological parameters (height of plant) and performance index (PIabs). Indeed, a striking correlation between the height of the plant and the performance index was observed. It was noticed that, plants showing maximum PIabs (treated with 500 ppm Mg(OH)₂NPs) demonstrated maximum height among all plants treated with various concentrations.

The significant plant growth promotion efficacy of Mg(OH)₂NPs with specific concentration may be due to the fact that nanoparticles may access the fortification in plant at nano level because of its more penetration capacity as compared to bulk materials. The results thus obtained revealed that nanoparticles are effective in very low dose i.e. at ppm level.

3.5 Detection of NPs uptake by plants using atomic absorption spectrophotometer (AAS) analysis

In vitro and in vivo grown Z. mays plants were analyzed by AAS, which is a simple and selective method for determination of total contents and speciation analysis of metals. This study was performed to determine the concentration of Mg in treated plants and to find out their correlation with enhancement in plant growth.

In the AAS analysis of leaves and roots of in vitro grown plants, it was found that, seedlings grown on MS medium supplemented with 500 ppm Mg(OH)₂NPs showed maximum Mg content in leaves (131.45 mg/kg), whereas seedlings treated with 200 ppm Mg(OH)₂NPs and control seedlings showed 94.58 mg/kg and 38.94 mg/kg of Mg contents respectively. Moreover, roots of seedlings treated with 500 ppm Mg(OH)₂NPs demonstrated maximum Mg contents (103.52 mg/kg) followed by that treated with 200 ppm Mg(OH)₂NPs (89.20 mg/kg) and control (34.32 mg/kg).

Similarly, in case of in vivo grown plants, among all the nanoparticles’ treatment, plants treated with 500 ppm of Mg(OH)₂NPs demonstrated higher contents of Mg in leaves (132.58 mg/kg) and roots (114.58 mg/kg). However, Mg contents in leaves and roots for plants treated with 100 ppm Mg(OH)₂NPs (93.69 and 78.96 mg/kg), 200 ppm Mg(OH)₂NPs (96.98 and 89.69 mg/kg), 1000 ppm Mg(OH)₂NPs (112.35 and 84.58 mg/kg) and control (59.80 and 39.79 mg/kg) were reported, respectively.

It was observed that the translocation of the nanoparticles was dependent on their concentration and different plants parts. The findings reported in the present study showed the resemblance with the observations of Ramos et al. (2002) and Gong et al. (2017) who demonstrated uptake of Cd in different plants. Although in the present study, further subcellular distribution of Mg in other cellular organelles was not analyzed. However, available report suggests that maximum uptake of such metals was observed in cell wall followed by protoplast and chloroplast (Ramos et al., 2002).

Generally, nanoparticles are applied to the plants by two methods i.e. in the form of foliar spray (Torabian et al., 2016) or inoculated to roots of plants. In foliar application, the nanoparticles penetrate inside the plant cells through the organs and tissues such as cuticles, trichomes, stomata and hydathodes. However, when nanoparticles are inoculated near the root, they enter into plant cells through the root tips, lateral roots, root hair, root wounds and root junctions (Nair et al., 2010; Wang et al., 2016). Although, the nanoparticles enter through plant parts as discussed above, there are various supportive molecules and mechanisms such as carrier proteins and other chemicals, pores on surface of cells, aquaporins, ion channels and endocytosis facilitate easy entry of nanoparticles (Gong et al., 2017). Moreover, it was proposed that adsorption, translocation and accumulation of nanoparticles depend on types of plant and also on size, type, chemical composition and stability of nanoparticles (Rico et al., 2011).

There are some studies which support that the effect of nanoparticles was actually due to their respective ions. Nanoparticles dissociate in water and soil and the ions released from them enter into plants. The rate of dissociation of nanoparticles is dependent on their size and the nanoparticles have ability to release the ions rapidly as compared to its bulk counterpart. The most important conclusion, which could be drawn from analyzing the absorption data, is that Mg concentration increases in leaf and roots of experimental plants, which proves the transport of nanoparticles in almost all over the plant region.

3.6 Estimation of chlorophyll content in nanoparticles treated Z. mays plants

Chlorophyll (Chl) content present in the plants can directly determine their photosynthetic potential and primary production. In addition, chl also gives an indirect estimation of the nutrient status because presence of nitrogen is directed to chlorophyll and we know that nitrogen is one of the most important components required for plant growth (Argenta et al., 2004). Furthermore, leaf chl content is closely related to plant stress. We found that plants treated with concentration of nanoparticles showed higher content of Chl a and b as compared to the control plants. It was observed that plants treated with 500 ppm Mg(OH)₂NPs showed higher Chl a (23.64 mg/g of fresh weight) and Chl b (34.46 mg/g of fresh weight). However, Chl a and Chl b contents for plants treated with 100 ppm Mg(OH)₂NPs (19.63 and 31.25 mg/g of fresh weight), 200 ppm Mg(OH)₂NPs (22.01 and 30.69 mg/g of fresh weight), 1000 ppm Mg(OH)₂NPs (22.21 and 29.36 mg/g of fresh weight) and for control plants these contents were 19.04 and 24.08 mg/g of fresh weight respectively. The increase in chlorophyll content of treated plants showed the maximum photosynthesis capacity, which is also reflected from other physiological and morphological characteristics of plants.