Introduction

Currently agriculture faces challenges caused by climate change, such as irregular rainfall, extreme temperatures and soil degradation, which can induce stress in crops and decrease productivity. Corn cultivation is an essential part of food systems, and finding effective ways to counteract the adverse effects of environmental variations is a primary task (Grzanka et al., 2021). To mitigate these adverse effects, promoting the natural resilience system of plants, which constitutes the central element of biostimulation and is achieved through the use of biostimulants, has been favored (Sariñana-Aldaco et al., 2022).

A biostimulant is any substance or microorganism applied to plants with the aim of improving their nutritional efficiency, tolerance to abiotic stress or quality traits, regardless of their nutrient content (du Jardin, 2015). Pregermination biostimulation or seed priming is considered a technique for improving metabolic processes prior to germination and increases the germination rate, seedling strength and crop yield, both in the presence and absence of biotic and abiotic stress (Rhaman et al., 2020).

Iodine is a beneficial nutrient for plants and it is also considered a powerful biostimulant that at low concentrations shows positive effects on plants growth (García-Fuentes et al., 2022). Iodine is applied as iodide or iodate (Medrano-Macías et al., 2016), associated with polymers such as chitosan (Dávila-Rangel et al., 2020), or in the form of low molecular weight organic molecules containing iodine (Krzemińska et al., 2023). In addition, it has been reported that it specifically regulates the expression of several genes, which are mainly involved in the defense response of plants, which suggests that iodine can protect plants against biotic and abiotic stress (Kiferle et al., 2021).

In recent years, the use of micro- or nanomaterials as sources of fertilizers or biostimulants has been studied, revealing greater efficiency and effectiveness compared to conventional materials due to the improvement of some of their physicochemical characteristics, explained by their high area/volume ratio. The micro- and nanoforms of different nutrients, biostimulants and biocides have been tested, and the results are encouraging and applicable in terms of increasing agricultural productivity (Cartwright et al., 2020; Gudkov et al., 2020; Lira-Saldivar et al., 2018). To date, there are no reports about the use of iodine in micrometric or nanometric forms, which are forms of the element that, compared to ionic forms, could be more stable for use in the soil, in nutrient solutions or by spraying, and they may be more easily absorbed by plant structures (Morales-Díaz et al., 2017).

Based on the above, this study aimed to: 1) synthesize and characterize polymeric particles functionalized with submicron iodine (PI), 2) evaluate the impact of pretreatment of corn seeds with the PI on the germination rate and growth of the seedlings, and 3) quantify the iodine in the PI and in the seedlings.

Materials and methods

This study was conducted in the Plant Physiology Laboratory of the Horticulture Department of Antonio Narro Autonomous Agrarian University (UAAAN). For the germination of seeds and the growth of the seedlings, a growth chamber with a temperature of 28 °C was used. The plants were illuminated with LED lamps containing white light and a photosynthetic irradiance of 90 µmol∙m^-2∙s^-1. The illumination period was 12 h per day.

Synthesis and characterization of iodinated particles (PI)

A mixture of 1 mmol of sodium periodate (NaIO₄) and 1 mmol of iohexol (C₁₉H₂₆I₃N₃O₉) was added to 200 mL of deionized water. The reaction was carried out using a 1:1 M ratio in a beaker with magnetic stirring for 30 min at room temperature. After this time, the sample was frozen for subsequent lyophilization in a liophilizer (A65312906, Labconco™, Czech Republic) for 24 h. Then 10 mL of distilled water was added to the obtained residue, and the mixture was polymerized with 0.5 mmol of carbohydrazide. A mixture of 0.002 mmol of amino polyethylene glycol (PEG) was added, after which the mixture was allowed to react for 24 h without stirring at room temperature. To stop the reaction, 1.6 mmol of sodium borohydride was added, and the mixture was center to react for 3 h at room temperature. Afterwards, the mixture was frozen overnight and lyophilized again for 10 h to obtain the iodinated particles (Hainfeld et al., 2018). The reaction products were characterized by infrared spectroscopy using an FTIR spectrophotometer (Thermo Fisher Scientific, USA) in triplicate with a scan of 650 to 4,000 cm^-1 and by proton nuclear magnetic resonance (¹H-NMR) spectroscopy in a spectrometer with a 5 mm multinuclear BBI-decoupling probe with Z grad (HD 400 MHz, Bruker Avance III™, USA), using D₂O as a solvent. The average diameter in number (Dn) of the iodinated particles was determined via dynamic light scattering (Nano-ZS90, Malvern Panalytica, United Kingdom) equipment at 25 °C and a measurement angle of 90°.

Iodine extraction method adapted from alkaline ash

A 500 mg sample of the synthesized PI was weighed, as well as the previously ground dry plant tissue (root and plumule) of the corn seedlings, from each repetition and treatment. Next, each sample was placed in a crucible, and then 2 mL of KOH at 2 M and 1 mL of KNO₃ were added at 2 M. Once the reagents had been added to the sample in the crucible, it was predigested in an oven at 100 (C for 2 h under a fume hood. Subsequently, the crucibles were placed in a muffle furnace at 580 °C for 3 h. After this time, the mixture was allowed to cool to room temperature, and the ashes were transferred to Eppendorf tubes for extraction with 2 mL of KOH at 2 mM. The tube samples were then centrifuged at 15,231 × g for 15 min in a centrifuge (R Ediso, Labnet Prism™, USA). The supernatants were collected with special care, from which 1 mL was taken and ground in a 10 mL flask with KOH at 2 M. The samples were subsequently transferred to conical tubes for later analysis via an inductively coupled plasma optical emission spectrometer (ICP‒OES, Agilent 725, USA) (Fischer et al., 1986).

Biological material

The hybrid corn seed used was Francisco Márquez, which was collected during the autumn-winter cycle and provided by the Mexican Institute of Maize of the UAAAN. The germination rate of this batch of seeds was 100 %.

Germination and growth test

For the germination test, four concentrations of PI were used (5, 10, 20, and 50 mg∙L^-1), and 10 mL of each solution was used for the imbibition of 50 seeds per treatment for 24 h at a temperature of 28 °C. After the indicated time, the seeds were extracted from the solution, and 10 seeds were placed in each of the five Petri dishes for each treatment in the growth chamber at 28 °C. A piece of filter paper was placed in each Petri dish that was constantly moistened with distilled water to ensure that the seeds were in a medium with water available. To determine the germination speed, the number of germinated seeds was visually counted every 12 h for 7 days. A seed was considered germinated when it showed a visible radicle of at least 1 mm long. After 7 days, the plants were collected, and the fresh weight of the roots and plumules of each plant was determined using a scale with a precision of 0.01 g. Subsequently, the plants were dried in a dehydrating oven at 70 °C for a period of 72 h to obtain the dry weight.

Statistical analysis

The experimental design used was completely randomized, with five treatments and five repetitions. The Levene test was carried out to verify the homogeneity of variance, and the Shapiro-Wilk test was performed to check for normality. The variables were analyzed by means of variance analysis and multiple range tests (Tukey, P ≤ 0.05). For the data that were not normally distributed, a nonparametric analysis of variance was performed with the Kruskal-Wallis test using InfoStat version 2020 statistical software.

Results and discussion

Characterization of iodinated particles (PI)

Figure 1 shows the FTIR spectrum of the PI, as well as its expected general structure, in which aromatic groups containing iodine atoms can be observed and linked together by chains from the crosslinking agent, as well as the presence of polyethylene glycol (PEG) as a coating for PI. Additionally, Figure 1 notes the most representative bands that can be assigned to the infrared spectrum of the PI, in which those attributed to the carbonyl groups, amides and carbon‒carbon double bonds of the aromatic groups stand out.

Figure 2 shows the ¹H-NMR spectrum of the water-soluble fraction of the PI. In the spectrum, some important signals can be assigned to functional groups of the expected structure of the PI (Figure 2b). For example, the presence of PEG as a coating was confirmed by the signal at ( 3.6 ppm, which was assigned to an ether group; signals were also observed at approximately ( 8.5 ppm, attributed to the amide groups, and at ( 1.8 ppm, associated with the acetate groups of the structure.

Figure 3 shows the particle size distribution curve determined by dynamic light scattering. The average diameter in number (Dn) of the PI obtained for this analysis was 217 nm, with a polydispersity of 0.278, which is why these particles were called submicrons. The shape of the curve is monomodal and narrow.

Germination and growth test

Once the PIs were characterized, they were tested as a biostimulant for the germination and vigor of corn plants. The germination percentage did not significantly differ between the treatments and controls. This is positive, since PIs do not negatively interfere with the seed germination process; however, at the appropriate concentration they accelerate germination. It should be noted that all treatments began to germinate 12 h after the plants were placed in Petri dishes (Figure 4). As a function of time, there were statistically significant differences at 12 h and later at 36 h between the treatments, reaching the final germination percentage at 48 h (Table 1). It is important to mention that seed pretreatment has the potential to improve germination and seed establishment by intiating germination metabolism without radicle protrusion (Adhikari et al., 2022). Seed germination is one of the most vulnerable processes in a plant’s life cycle, since it favors the healthy and vigorous development of seedlings, which is greatly affected by internal and external changes; in addition, it has been shown that low crop productivity is related to uneven seed germination (Salehi et al., 2023).

Table 1. Effects of different iodinated particle concentrations on the percentage of germinated corn seeds.

In terms of the growth curve of the root system, the emerging of the radicle began on the second day after sowing in all treatments, and the growth of the main root progressed continuously but increased in intensity throughout its development. In addition, when analyzing the growth pattern of this variable (Figure 5), a small increase was observed on the third and fourth days, during which the growth pattern increased considerably beginning on the fifth day of the experiment. Similarly, the plumule stage (Figure 6) occurred on the third day after sowing, during which the height accelerated from the fifth day in all treatments. For both variables, treatment five presented better development than the other treatments, including the control.

The results shows that treatment five (50 mg∙L^-1 PI) significantly increased the height and dry weight of the plumules, as well as the dry weight of the roots, compared to the control and other treatments Therefore, seedling growth was stimulated by the addition of 50 mg∙L^-1 PI. According to the data reported in Table 2, radicle length and fresh weight did not significantly differ from the control.

Table 2. Response of the variables evaluated at different concentrations of iodinated particle.

These results are consistent with those of previous rice cultivation experiments in which zinc oxide nanoparticles were used at different concentrations (0, 5, 10, 15, 25, and 50 ppm), where increases in height and the fresh and dry weights of the plants were obtained (Waqas-Mazhar et al., 2022). In the same way, in studies carried out with corn seeds, using different pretreatment techniques with different solutions significantly improved the performance of the corn plant by increasing the germination rate of the seeds, the biomass of the seedlings and the yield of the seeds. However, the response varies with solution and concentrations (Tian et al., 2014). The above results show that seed pretreatment stimulated several signaling cascades during the early growth phase, producing faster and more efficient defense responses in the crop, thus tending to favor better synchronization of crop growth by increasing both fresh and dry biomass (Adhikari et al., 2022).

Seeing these results, and regardless of the possibility of iodine toxicity in plants, it has been described that low iodine concentrations increase yield, biomass production and qualitative parameters of plants (Duborská et al., 2018; Medrano-Macías et al., 2016). Thus, the different concentrations of PI used can be considered to be appropriate since they significantly improved the growth and development of the plants, by strengthening their immune system and protecting them from oxidative stress since they act as a form of controlled release of iodine in the plant, which improves the absorption of the nutrient by the plants and this, in turn, has a direct impact on the growth variables, by stimulating the regulators of cell division, accelerating the production of photosynthetic pigments and playing a role in the enlargement of the roots (Rahman et al., 2023).

The favorable results obtained are largely due to the absorption capacity of iodine in the cells of the root, leaf or stem. This implies that the concentration of gradients is the third force in transporting iodine, migrating it from the root to the stem and leaves by diffusion, in addition to transpiration and root pressure (both advection movements); however, the absorption capacity is different for different plant species (Weng et al., 2013). Iodine transport from roots to shoots could be regulated by transporters (Kato et al., 2013). Iodine has been reported to potentially play a nutritional role in plants, as the application of small amounts (micromolar) enhances their growth and development. This treatment not only stimulates biomass production but also promotes early flowering. Such effects cannot be replicated with similar halogens, such as bromine, due to iodine's role as a structural component of various proteins (Kiferle et al., 2021). Recent studies consider that iodine may not be an essential element, but it is beneficial like silicon (Si), selenium (Se), sodium (Na) and others (Medrano-Macías et al., 2021; Nascimento et al., 2022).

Iodine content

The iodine content in the synthesized PI was 1.67 %. Although there were no differences in the content of I among the treatments, the highest concentration (205.96 mg∙kg^-1 dry weight) was found in the corn plants subjected to 50 mg∙L^-1 (Table 3). The absence of differences in the iodine content among the treatments is likely the result of contamination when the plants were grown in the same growth chamber, possibly caused by the volatilization of iodine from the seedlings (Gonzali et al., 2017). This assumption is based on the fact that the iodine concentrations in the biomass of the plants are well above the normal values found in the plants, especially for cereal grains for which maximum values of 128 (g∙kg^-1 are reported (Dávila-Rangel et al., 2019). Therefore, these results are promising since, in addition to its biostimulant impact, PI could be used as a material for the biofortification of crops to increase the daily intake of iodine (Duborská et al., 2020). With the results obtained when iodine microparticles were used as agents for seed priming, we agree with the findings of other authors who point to iodine as a biostimulant nutrient (Blasco et al., 2008).

Table 3. Iodine content in the biomass of the plants in the different treatment.

The results obtained suggest that the application of these particles functionalized with iodine represent a promising system to be used in the formulation of new crop biofortification strategies, since they positively influenced the development of corn seedlings, as well as the absorption and greater amassing of iodine. However, more research is needed to verify whether the iodine-functionalized submicron particles produced can show the same positive effects in other plant species.

Conclusions

Iodine-functionalized submicrometric particles with an average diameter of 217 nm were synthesized. With different concentrations of these particles as a pretreatment for corn seeds, no effects were observed on germination; however, a biostimulant effect was evident on the growth and development of seedlings, as the 50 mg∙L^-1 treatment significantly increased biomass. Finally, the presence of iodine in the dry biomass of the seedlings and 1.67 % iodine in the synthesized particles was found.