Effect of iron nanoparticles on seed germination and early seedling growth of finger millet (Eleusine coracana L.)

Introduction

Iron is the fourth most abundant element in the Earth's crust. However, its actual availability to plants remains limited because it readily precipitates into insoluble hydroxides under natural soil conditions. Aeration and drainage conditions have a significant influence on the chemical speciation of iron in soils. Iron is an essential micronutrient for better growth and development of plants. In the plants it is required not only for energy production but also for sulfate and nitrate reduction. In chlorophyll, iron acts as a key precursor, iron is required as an active site of glutamyl-tRNA reductase, an enzyme for the formation of 5-aminolevulinic acid. Due to limited mobility of iron within plant tissue, the younger leaves show interveinal chlorosis. Iron deficiency affects physiological functions of plants and thus normal growth and development. Not only for plants, iron plays an important role in human health, as it's essential for hemoglobin synthesis, biochemical pathways, and electron transport reactions (Olivares et al., 1999) [4] . Plants get their iron needs from the soil and the beneficial bacteria in it. In recent years, application of nanotechnology in the agriculture area has been highlighted as a possible alternative to conventional fertilizers. Nanoparticles (NPs) are particles with sizes typically ranging from 1 to 100 nm. NPs help in plant growth and development due to their physiochemical properties, like high surface area to volume ratio (Reddy and Chhabra, 2022) [7] . Several nanofertilizers, like nano urea, nano DAP, nano zinc, nano nitrogen, etc., are available in the market (Mondal, 2022) [3] .

Iron Nanoparticles (INPs) help in increasing nutrient availability, uptake, translocation and physiological response in plants. INPs also help in enzyme activity, metabolic activation, water uptake and nutrient availability. NPs also enhance micronutrient uptake, stimulating the biosynthesis of antioxidant and secondary metabolites. Finger millet (Eleusine coracana L), which is commonly known as ragi or nagli, is a nutritionally rich millet crop commonly cultivated in semi-arid and tropical regions. Finger millet is the fourth largest crop among millets and the sixth largest crop among cereals. Due to finger millet's nutritional composition and capability to grow under drought conditions and marginal soil conditions, it is highly valuable. Finger millet is particularly notable for its significantly high iron content, and it serves as an important dietary source. Apart from that, it is also a rich source of calcium, protein, fiber and other minerals that contribute to its nutritional and health-promoting properties. For diabetic patients, finger millet helps to regulate blood sugar level due to its low glycemic index. Its anti-inflammatory and antioxidant compounds help in improving digestive and metabolic health. Finger millet is referred to as "Nutricereal" due to its superior nutritional quality, and finger millet plays an important role in preventing malnutrition and micronutrient deficiencies, especially iron deficiency in anaemia, which may contribute to improving public health by helping address micronutrient deficiencies. In particular, understanding the response of finger millet seeds to different INP concentrations can help identify concentrations that promote germination and early seedling vigour while avoiding potential inhibitory effects. Therefore, the present study was undertaken to evaluate the effects of different concentrations of iron nanoparticles on seed germination and early seedling growth of finger millet (Eleusine coracana L.) and to identify a concentration range favourable for seedling establishment.

Materials and methods

Plant material and experimental conditions

For the seed germination experiment, finger millet seeds were used (variety: gira). Healthy and undamaged seeds were used for further study. The experiment was conducted at laboratory-controlled conditions to check the response of different concentrations of INPs on finger millet seeds.

Iron nanoparticle preparation

INPs were synthesized by the co-precipitation method as described by Palaniswamy et al. (2013) [5] . To begin with, equal volumes of 1M ferric chloride (FeCl 3 ) and 1.5 M ferrous sulfate heptahydrate (FeSO 4 •7H₂O) solutions were mixed in a clean glass beaker and placed on a magnetic stirrer under continuous and vigorous stirring. To the prepared mixture, 3 M sodium hydroxide (NaOH) solution was added at the rate of one drop per second. The addition of NaOH was continued until the solution reached a pH between 10 and 11. At that point a visible color change occurred and a formation of a black precipitate. The precipitate obtained during synthesis was carefully washed with deionized water to remove any residual salts and impurities from the solution. Using the magnetic decantation method, separation of the black precipitate was achieved, which ensured effective collection of the nanoparticles. The collection material was subsequently dried in a hot air oven at 150 °C for 2-3 hr. The resulting dried product was identified as iron nanoparticles, which were subsequently utilized for further experimental investigations.

Experimental design and INPs treatments

The experiment was conducted in a completely randomized design (CRD) with seven treatments and three replications (T1-T7), including the untreated control. Each treatment was maintained in three replications, with 10 seeds per Petri plate. Thus, each treatment consisted of 30 seeds. [11] . To find LD 50 , seeds were grown at different INP concentrations (0.05, 0.1,1,2,3 and 4 mg/10ml) in sterile germination plates and % seed germination was calculated. The plates were incubated under the experimental laboratory conditions used for the study. Moisture was maintained throughout the experimental period, and Hoagland solution was added to maintain nutrition to ensure adequate conditions for seed germination. Germination observations were recorded for 7 days. Seed germination was recorded when visible radicle emergence was observed.

Determination of germination percentage

Germination percentage was calculated based on numbers of seeds germinated relative to the numbers of total seeds (Akshitha et al. 2026) [1] . Ten seeds were used per replication, with three replications for each treatment, giving a total of 30 seeds per treatment. Germination percentage was calculated using the formula: Germination (%) = (Number of germinated seeds / Total number of seeds) × 100

Measurement of seedling growth

Seedlings were carefully removed from Petri plates after the experimental period. For measurement of root and shoot length of each seedling using a scale and expression in centimeters (cm). Root length was measured from the point of root emergence to the tip of the primary root, while shoot length was measured from the seed base to the tip of the shoot. The recorded root and shoot measurements were used to determine the effect of INP treatment on early seedling development.

Seed vigour Index

Seed Vigour Index (SVI) is a key indicator of overall seedling performance, as it integrates germination percentage with seedling length (Akshitha et al. 2026) [1] . The seed Vigour Index was calculated using the formula:

Seed Vigour Index = Germination (%) × Seedling length (cm) [9] reported that the effect of INPs increased germination and early seedling growth at optimal concentrations of INPs. At higher concentrations shows an inhibitory response.

Results and discussion

Effect of iron nanoparticles on root length

The effect of INPs on root length shows a clear dosedependent response. The untreated control (T1) recorded a 3.58 cm root length. Among the other treatments treated with different INP concentrations, T3 showed the highest root length (3.52 cm) other than the control, followed by T2 (3.36 cm). When INP concentration increases, with root length decreasing to 2.21 cm in T4, 2.31 cm in T5, and 1.57 in T6 and lowest root length was found in T7 (0.68 cm). The critical difference (CD) at 5% was 0.78 cm, showing significant variation among the treatments. Root growth was comparatively less affected at lower INP concentrations, while higher concentrations markedly inhibited root elongation. The responses of seed grmination and root development exhibited a similar decreasing trend with increasing INP concentration in the present study. Germination decreased from 100.00% in the control T1 to 23.33% at T7, while root length decreased from 3.58 cm in the control to 0.68 cm at the highest concentration. Among the INP treatments, T3 recorded the highest root length (3.52 cm), which was close to the control, followed by T2 with 3.36 cm. At higher concentrations, root length declined to 2.21, 2.31, 1.57 and 0.68 cm in T4, T5, T6 and T7, respectively. Thus, unlike the contrasting response described previously, the present data indicate that increasing INP concentration adversely affected both seed germination and root elongation. The pronounced reduction in root length at T6 and T7, together with the corresponding decline in germination percentage, suggests that higher INP concentrations exerted an inhibitory effect on early seedling development. Overall, T3 appeared to be the most favourable INP concentration among the treated groups for maintaining root growth.

Statistically, root length differed significantly among treatments, with a S.Em. of ±0.25 cm, CD (5%) of 0.78 cm, and CV of 17.84%. The differences between treatments exceeding the CD value were considered significant. T1 and T3 recorded comparatively higher root lengths, whereas T6 and T7 showed significantly lower root growth.

Effect of iron nanoparticles on shoot length

The effect of INPs on shoot length shows dose-dependent manner. T1 (untreated control) shows the highest shoot length of 1.74 cm, followed by T3 with 1.70 cm and T2 with 1.61 cm. With increasing INPs concentration, shoot length decreases from T4 (1.35 cm) to T7 (0.357 cm). Thus, higher INP concentrations, particularly T6 and T7 substantially inhibited shoot elongation. Among all the treatments, T3 showed shoot growth closest to the control. Statistically, shoot length varied significantly among the treatments, with an S.Em. of ±0.13 cm, CD (5%) of 0.41 cm, and CV of 17.65%. Treatment differences exceeding the CD value were considered significant. Overall, shoot growth declined with increasing INP concentration, and the highest inhibition was recorded under T7.

Effect of iron nanoparticles on total seedling growth

The effect of INPs on total seedling length shows the dosedependent manner, with increasing IPNs concentration decreasing total seedling length. The highest seedling length was observed in T1 (5.31 cm), followed by T3 (5.22 cm), and T2 (4.97 cm). At higher concentrations, total seedling length decreased in T5 (3.68 cm), T4 (3.56 cm), T6 (2.67 cm) and T7 (1.03 cm). Thus, increasing INP concentration, particularly T6 and T7, substantially reduced overall seedling growth. Among the INP treatments, T3 maintained total seedling length closest to the control. The response of total seedling length varied with INP concentration and should be interpreted alongside germination percentage. In this study, the highest total seedling length was recorded in the control (T1) at 5.31 cm, followed closely by T3 at 5.22 cm and T2 at 4.97 cm. At the higher concentration of INPs, seedling length decreased from in T4 (3.56 cm), T5 (3.68 cm), T6 (2.67 cm) and T7 (1.03 cm). This decline was followed by a significant decrease in germination from T1 (100 %) to T7 (23.33 %). Therefore, total seedling length alone should not be considered an indicator of overall treatment performance. The combined response of germination and seedling growth indicates that higher INP concentrations, particularly T6 and T7, adversely affected early seedling establishment and development, whereas T3 maintained seedling growth close to the control with a relatively high germination percentage of 93.33%. Total seedling length showed a statistically significant difference within all the treatments, with S. Em ± (0.34 cm), CD at 5% (1.04 cm) and CV (15.49 %). Differences greater than the CD value were considered significant. Overall, results indicate that the higher concentration of INPs showed an inhibitory response to seedling development, with the lowest seedling growth observed at T7. The present finding aligns with Plaksenkova et al. (2019) [6] and Shankramma et al. (2016) [10] , which shows that INPs increased germination, root length and shoot length and shows dose dependent response. Mohan et al. (2025) [2] investigated the effect of INPs on okra. After 10 days at 0.5 mM, INPs increased seedling root length by 2.9 cm and shoot length by 9.2 cm. The findings of the present study are consistent with previous reports demonstrating that the effect of INPs on seed germination, seedling length and SVI. Sarma and Prakash (2019) [8] and Triveni et al. (2021) [12] reported INPs, enhance seed germination and root and shoot length, at lower concentration, whereas higher concentrations reduced germination and seedling growth, indicating a dosedependent response.

Acknowledgement

Acknowledgement:The authors gratefully acknowledge Navsari Agricultural University for providing the financial support required to conduct this research.

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