Zinc Oxide Nano Priming: A Cutting-edge Strategy for Mung bean (Vigna radiata L.) Seed Bio-stimulation

The global food supply faces numerous challenges due to the increasing global population. It is predicted that the human population will hit 9.7 billion by 2050. Due to this population increase, the demand for food will increase between 59% to 98% (Haris et al., 2023). Another challenge is providing nutritious food. An important legume, mung bean, is recognized for its medicinal and nutritional values (Liang et al., 2022). It is rich in protein, fiber, vitamins and minerals and free from gluten and cholesterol.
       
Mung bean (Vigna radiata L., Family: Fabaceae), popularly called green gram or moong bean, has been consumed for about 3,500 years (Junjariya et al., 2024). It has been cultivated mainly in India and introduced worldwide (Choudhary et al., 2025). This Mung bean sprouted food is gaining demand as a therapeutic food (Geng et al., 2022). Because of its dual benefits in medicine and food, it is accepted as a Medicine Food Homology (MFH). In a few restaurants, sprouted Mung bean is sometimes served as a main or side dish (Salvador et al., 2021). It is also produced as value-added products (Naik et al., 2020). Mung bean cultivation faces enormous challenges such as poor seed quality, weeds and pests, degradation of soil, inadequate farming practices, quality issues and postharvest wastage (Dikr et al., 2023).
       
Nanomaterials can be used as fertilizer that prevents nutrients from prematurely converting into gaseous or chemical form (Verma et al., 2023). The commonly utilized NPs were zinc (Zn), titanium (Ti), iron (Fe), copper (Cu), manganese (Mn) and aluminum (Al) (Maity et al., 2022). Reports indicate that ZnO NPs priming in Maize seeds increases nutrient, antioxidant enzymes, biomass yield, growth rate and photosynthetic performance under adverse environments and reduces Malondialdehyde (MDA) and Shoot Reactive Oxygen Species (ROS) accumulation (Salam et al., 2022). The use of ZnO NPs Priming in cucumber plants improved physical parameters such as germination rate, the height of the plant, leaf number and area, flower yield and its fruit length, weight, diameter, color, firmness and chemical parameters such as total soluble solids, ascorbic acid, titratable acidity (Nisar et al., 2022). ZnO NPs priming in mung bean seeds under salinity conditions enhanced germination percent, physical parameters,  also increased antioxidant enzyme activity, proline content, specifically superoxide dismutase (SOD) and peroxidase (POX) (Sherpa et al., 2024). The ZnO NPs priming in seeds of tomatoes under a stressful environment increases germination, chlorophyll, enzyme-mediated and non-enzyme mediated antioxidant protective mechanism and adjusts osmotic conditions (Ghosh et al., 2024). The ZnO seed priming stimulated germination and increased fruit number, density, diameter and phytochemicals were noticed in Bitter gourd seeds (Mazhar et al., 2024). Germination and total phenol content were improved by ZnO NPs priming in sunflower seeds (Al-Sudani et al., 2024). The ZnO NPs priming in rapeseed boosts germination and vigor index (VI), increases the physical parameters, absorption of nutrients and antioxidants and adjusts osmotic protection leading to proline, soluble protein and sugar increase. Reduces ROS, degradation of chlorophyll and biosynthesis pigment under salinity (El-Badri et al., 2021).
               
In the present work, mung bean seeds were selected due to the enormous benefits mentioned. ZnO NPs were used to prime the seeds due to their popularity, easy availability and low price. The ZnO NPs are synthesized through a simple combustion method. Various techniques like UV visible spectroscopy, XRD, RAMAN and FE SEM were used to characterize the synthesized ZnO NPs. Priming the seed is a low-cost, feasible technique and can advance germination in the early growth stage of plants in adverse environments. Mung bean seeds were primed at different ZnO NPs concentrations. After seed germination, the physical and phytochemical parameters of the sprouts were examined and a comparison was carried out between the control group seeds and primed seeds.

Chemicals
 
All Chemicals used were analytical-grade and were not further modified, with ultra-pure water used in all procedures.
 
Synthesis of ZnO NPs
 
The self-propagating high-temperature synthesis (SHS) technique was employed to prepare ZnO NPs at the Nanomaterials Synthesis Laboratory, Noorul Islam Centre for Higher Education, Kumaracoil. In a typical experiment, zinc nitrate hexahydrate (0.2 g) and ascorbic acid (0.1 g) were mixed in an agate mortar. The blend was placed into an alumina crucible and heated in a muffle furnace at 500^oC in stagnant air for 3 hours to yield the ZnO  NPs as shown in (Fig 1) (Kumar et al., 2011).


 
Characterization of nanoparticles
 
The ZnO NPs were characterized for their optical properties, crystalline nature, vibrational modes and morphology. The optical properties were analyzed using a Systronic M2202 double-beam UV-visible spectrophotometer. ZnO NPs crystalline structure was studied through X-ray diffraction utilizing the BRUKER D2 Phaser instrument. Vibrational modes of the ZnO NPs were obtained using a RAMAN spectrophotometer (EZRaman, N785, Enwave Optronics, USA). Additionally, the ZnO NPs morphology was investigated with FESEM-SUPRA55, manufactured by Carl Zeiss, Germany.
 
Seed priming experiment in mung bean (Vigna radiata) seeds
 
The mung bean seeds were washed in distilled water to remove surface dust and treated with 70 % ethanol for 2 minutes to remove surface microbes. Afterward, a 15-minute rinsing with 1% sodium hypochlorite was conducted to eradicate any bacteria or fungi contamination on its surface and subjected to 5 consecutive washes with ultrapure water (Tadayon et al., 2023). Then, priming with ZnO NPs of various concentrations and the control group were treated with distilled water in a closed environment at room temperature for 24 hours. After that, seeds from each treatment were placed separately in Petri dishes  above sterile tissue paper and 5 mL of distilled water was added (Masilamani et al., 2025). These seeds were kept undisturbed for a week to grow within the Petri dish. The entire experiment was carried out in triplicate at the Nano Applications and Research Laboratory, Noorul Islam Centre for Higher Education, Kumaracoil, from January 2024 to June 2024, under undisturbed, sterile conditions to avoid contamination.
 
Seedling growth parameters
 
After a week of germination, the shoot and root lengths and the dry and wet weights of the germinated mung bean seeds were measured.

Dry weight
 
The seeds were left to dry overnight inside an oven at 40^oC for 3 days (Anaemene et al., 2022). After complete drying, the dry biomass (DWs) was measured.
 
Plant extract preparation
 
The dried plant (0.5 g) was extracted in a reflux condenser using ultra-pure water of 50 mL for 2 hours at 100^oC. The extracts were collected and stored at 4^oC (Bitwell et al., 2023).
 
Carbohydrate content
 
The total carbohydrate was estimated using the Anthrone method. Briefly, 2 % Anthrone reagent 4 mL was added to the extract of 0.1 mL and the solution was incubated for 15 min at 90^oC. This was cooled immediately after heating. The blank was prepared only with the Anthrone reagent and without the extract. The greenish-blue solution absorbance was recorded in a UV-vis spectrophotometer at 620 nm. The carbohydrate content was calculated using a calibration graph prepared using glucose as the stock solution (Plummer, 1990).
 
Protein content
 
The total protein present was estimated using Lowry’s method with Bovine Serum Albumin (BSA) serving as the standard. This method employs two types of reagents, Reagent 1 contains 0.4% NaOH and 2% Na₂CO₃ of 48 ml, 1% NaK tartrate and 0.5% CuSO₄.5H₂O. Reagent 2 contains 10% of the Folin-Ciocalteu reagent. To the 0.1 mL extract, reagent 1 of 4.5 mL was mixed and 10 min incubated. Subsequently, 0.5 mL of reagent 2 was added and incubated for 30 min. A blank was prepared with reagents 1 and 2 alone. The absorbance of the blue-colored solution was measured at 660 nm (Redmile-Gordon et al., 2013).
 
Total phenol content
 
The total phenolic content (TPC) present in the extract was estimated by using the Folin-Ciocalteu method, with gallic acid serving as the standard. In this procedure, 0.1 mL extract was added to Folin-Ciocalteu’s phenol reagent of 1.5 mL and left at room temperature for around 5 minutes. Subsequently, 20% sodium carbonate of 4 mL is added and incubated at room temperature for 30 minutes. Using a calibration graph TPC was estimated. The blue-colored solution absorbance was recorded at 765 nm (Isaac et al., 2012).

UV-vis spectroscopy result
 
The UV-visible wavelength range shown in (Fig 2 A) is between 355-400 nm. A strong absorption was measured at 367 nm, which is in accordance with previous research work done by a solvothermal approach and confirms the ZnO NPs (Singh et al., 2012).


       
The energy bandgap of the material was measured from Tauc’s plot at 3.1 eV, as shown in (Fig 2 B) calculated using equation (1).

XRD result
 
X-ray Diffraction (XRD) was used to characterize the ZnO NPs as shown in (Fig 3). The phase is found to be Hexagonal with JCPDS Card No. 36-1451, conformed XRD patterns of 500°C calcinated ZnO NPs. The reflections observed at 31.8° , 34.4° , 36.3° , 47.5° , 56.6° , 62.8°, 66.4°, 67.9°, 69.1°, 72.5°  and 76.9°, were indexed to planes as [100], [002], [101], [102], [110], [103], [200], [112], [201], [004], [202]. It states that ZnO NPs have no additional peaks and are highly pure. The average crystallite size (D) was calculated as 10 nm by using the Scherrer equation given below in Equation (2) (Jayswal and Moirangthem, 2018).

               
RAMAN result
 
The ZnO NPs Raman spectrum is shown in (Fig 4) measured under 785 nm red LASER excitation. The observed signal at 245 cm^-1, was due to interstitial zinc (Zni) affection (Song et al., 2019). The signal found at 403 cm^-1 resulting from A1 (TO) phonons oxygen-dominated polar mode (Abdelouhab et al., 2020). The remaining signals at 523 cm^-1, 549 cm^-1, 635 cm^-1, 688 cm^-1 were due to oxygen vacancy defect (Vo) (Song et al., 2019), longitudinal optical (LO) phonon modes of A1 and E1 modes (Khan et al., 2015), Second-order Raman scattering by LA(X) or LA(L) phonons and the combinational mode [LO (Γ) + TO(X)] (Milekhin et al., 2012), E1 (TO) mode (Lo et al., 2010).


 
FESEM image
 
The morphology of the ZnO NPs was examined using Scanning electron microscopy (SEM) illustrated below (Fig 5). SEM analysis further revealed a diverse range of particle sizes, spanning from 20 to 40 nanometers. This extensive size distribution shows the polydispersity inherent in the synthesized ZnO NPs.


 
Germination rate and seed vigor index
 
Germination was confirmed upon the emergence of a 2 mm radicle from the seed. After 1 week, all the germinated seedlings from each of the 3 replications, 5 were chosen and measured for both root and shoot lengths (centimeters) and the wet and dry weights (grams). The germination of mung bean seeds at different concentrations is illustrated in (Fig 6).


       
Various growth and development parameters were assessed, such as germination percentage (GP) Equation (3), seedling vegetation index (VI) Equation (4) and mean emergence time (MET) Equation (5). The results of these analyses are presented in Table 1 (Abdul-Baki et al., 1973).      

              

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The observed increase in the germination rate can be linked to the entry of NPs through seed coat absorption, the nature of the increased surface area to small size subsequently traversing by parenchymatous tissues, spacing enter cells easier. The positive germination percent indicates that accelerated amylase activity may improve simple sugar stock and lead to embryo viability. Additionally, the involvement of ROS during seed germination plays a crucial role in these processes (Setty et al., 2023).
       
In basic, the  quality of seeds was determined through 2 major parameters, GP and VI (Hussain et al., 2021). The simple calculation for determining VI is the percentage of germination multiplied by the shoot and root length. The results revealed that (3 ppm) showed a higher VI value of (3098.00) as compared with other treatments. A higher VI suggests greater vitality and potential for robust seedling growth (Roy et al., 2024). For higher VI, priming for 24 hours is found to be best (Muttaqin et al., 2019). Mung bean seed germination with ZnO NPs priming was dosage dependent and it inhibited faster growth and seedling traits (Sherpa et al., 2024).
 
Seedling growth assay
 
After growing the seeds for 7 days, measurements were taken for Wet weight (WW) and dry weight (DW), shoot length and root length. The results (Fig 7) showed that a higher material mass is yielded in terms of WW and DW when the ZnO concentration is at 5 ppm, compared with other treatments. The pivotal function in the pre-growth phase is supported by well-developed roots, which leads to the subsequent expansion of leaves by efficient nutrient and mineral absorption (Nakasha et al., 2014).


 
Carbohydrate content
 
The total carbohydrate levels in the control and NP-treated seedlings are shown in (Fig 8A). Zn is a crucial component in carbohydrate metabolism, contributing to improved and uniform seed germination. A decreased level of carbohydrate content at (5 ppm) than control was observed. The physiological discrepancy between ZnO NPs and control was because Zn²⁺ is slowly released from ZnO NPs. In lettuce seeds  ZnO NP treatment, decreased total carbohydrate content, because NPs trigger the early breakdown of carbohydrates and reduce overall carbohydrates (Rawashdeh et al., 2020). These findings are consistent with our study, as ZnO NPs promote faster growth through the rapid utilization of carbohydrates.


 
Protein content
 
The germinated seeds’ protein content was measured after 7 days and shown in (Fig 8B). Numerous studies have found that NPs mimic the antioxidant enzyme’s role and act as catalase (Wu et al., 2019). NP’s role in promoting the antioxidant defense mechanisms of plants, contributing to the resistance to survive in challenging environmental conditions (Rawashdeh et al., 2020). The total protein content result shows that the controlled seeds exhibit higher protein stability due to no externally induced stress. NPs-treated seeds experience a decline in protein levels due to mild stress but are still found non-toxic even at 5 ppm.
 
Phenol content
 
The NP-treated seedling’s Total Phenolic Content (TPC) is shown in (Fig 8C). The phenolic levels show an increasing trend in primed seeds compared with unprimed ones. Phenolic acids act as secondary metabolites of plants and are connected with biotic and abiotic plant responses to stress and dissolve in estimating the total activity of antioxidants. This improvement in TPC was particularly notable in treatment with 5 ppm ZnO NPs compared with other treatments. The increased phenolic contents may be indicative of a defense mechanism in overcoming potential NP-mediated phytotoxicity (Sorahinobar et al., 2023). The increase in phenolic production was found to be 1.3-fold compared to the control.

Mechanism of action
 
The mechanism behind ZnO NPs priming is illustrated in (Fig 9). Nanoscale materials possess diverse physicochemical properties, allowing them readily absorbed by seeds than bulk materials. Modifying surface chemical characteristics of nanomaterials may improve their molecular interaction with seedlings and reduce the waste of priming agents. Multiple studies have shown their capacity to hinder the development of both pathogenic and non-pathogenic bacteria and fungi. NPs’ notable properties allow for electron exchange and improve surface reactions in plant cells and tissues. Nano-priming induces nanopores, facilitating the absorption of water (Donia et al., 2023). The Zinc causes an increment in several cells and a larger size by producing auxin for the development of plants (Hussain et al., 2021). Nano-primed seeds, uptake water so that Indole-3-acetic acid (IAA) induces the hormones Abscisic acid (ABA)/Gibberellic acid (GA) that may convert starch into soluble sugar because of enhanced activation of α-amylase. This enabled the seed’s respiration response. Also, the hormones promote seed coat phenolics for passing nutrients into seeds and improve the seed germination and growth rate. But still, there was no clear internal mechanism reported on seed growth (Nile et al., 2022).

In agricultural practice, priming the seed is an alternative and easiest method for growing crops in stressful and normal conditions. The optimization of the seed priming technique improved crop performance, enhancing the productivity of plant crops and offering sustainable solutions for promoting overall agricultural productivity. Zn, a crucial micronutrient for plants, plays a vital role in various physiological functions. An adequate Zn supply in plants supports optimal growth and overall productivity in agricultural systems. Among NPs, ZnO NPs are the most extensively utilized, with several reports highlighting their beneficial effects on the ecosystem. This present study demonstrated the efficacy of ZnO NPs priming in improving biomass production in Mung bean seedlings compared to untreated seeds. Moreover, seed-priming with ZnO NPs enhances shoot height, root length and shoot biomass. The current findings indicate the effectiveness of ZnO NPs in improving seedling growth and development. Therefore, greater plant growth with NPs may result from better nutrient mobilization and stimulated microbial activity in the rhizosphere. Hence, nanotechnology is used to enhance germination and yield. In the future, research needs to be focused on practical field trial experiments.

This research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors.
 
Authors’ contributions
 
Jeyani Shalu; Carried out the experiments. Jeyani Shalu and Rimal Isaac; Co planned the experiments. Jeyani Shalu, Rimal Isaac and R. Revathi; Contributed to the interpretation of results and wrote the manuscripts.
 
Ethical approval
 
Not applicable.

The authors declare that there are no conflicts of interest related to this article.