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Article

Efficiency of Biofortification with Zn and Se in Soybean: Yield and Overall Mineral Content in Plant

by
Zdenko Lončarić
1,
Ivana Varga
1,*,
Franjo Nemet
1,
Katarina Perić
2,
Jurica Jović
1,
Vladimir Zebec
1,
Vladimir Ivezić
1,
Dario Iljkić
1,
Lucija Galić
1 and
Aleksandra Sudarić
2
1
Faculty of Agrobiotechnical Sciences Osijek, Josip Juraj Strossmayer University of Osijek, Vladimira Preloga 1, 31000 Osijek, Croatia
2
Agricultural Institute Osijek, Južno Predgrađe 17, 31000 Osijek, Croatia
*
Author to whom correspondence should be addressed.
Appl. Sci. 2024, 14(23), 11349; https://doi.org/10.3390/app142311349
Submission received: 7 November 2024 / Revised: 28 November 2024 / Accepted: 2 December 2024 / Published: 5 December 2024
(This article belongs to the Special Issue Crop Yield and Nutrient Use Efficiency)
Browse Figures
Versions Notes

Abstract

:
Since plant’s edible parts are one of the most important sources of nutrition, agronomic biofortification plays a huge role in overcoming mineral deficiency worldwide. The field-based research trial was set up in 2 years (2020 and 2021) with seven different treatments of foliar Zn and Se biofortification: 1. control (without Se or Zn solutions); 2. Se_1 treatment: 10 g/ha Se; 3. Se_2 treatment: 20 g/ha Se; 4. Se_3 treatment: 30 g/ha Se; 5. Zn_1 treatment: 3 kg/ha Zn; 6. Zn_2 treatment: 6 kg/ha Zn; 7. Se_3 Zn_2 treatment: 30 g/ha Se + 6 kg ha Zn. There were six soybean varieties of the 00 to I maturity group (Ika, Korana, Lucija, Sonja, Sunce, and Toma) included in the study, which originated from the Agricultural Institute Osijek, Croatia. After sampling the plants at the harvest, the macro- and micronutrient status in the grain, pods, leaves, and stems were determined, as well as nutrient removal by the plant. In general, biofortification treatment has a very significant influence (p < 0.001) on both Zn and Se accumulation in soybean grain and the removal of the elements within all above-ground organs. The highest increments of Zn in the soybean grain were determined at the Zn_2 treatment, which was 43% higher than the control treatment. The Toma variety accumulates the highest Zn in the grain (61.47 mg/kg), and the Lucija variety accumulates the highest Se (1070.71 µg/kg). The Se content in the soybean grain was the highest at the Se_3 treatment, where it was 53 times higher compared to the control. The linear regression showed that for each kg Zn and g Se applied, the grain status increased by 3.18 mg/kg and 338.71 µg/kg, respectively. The highest Zn nutrient use efficiency (NUE) of foliar biofortification for grain (2.6%) and vegetative mass (4.4%) was with 3 kg/ha (Zn_1). Generally, for all the Se treatments, it was found that the seed and vegetative mass yields of 4.0 t/ha have average Se NUE, around 38%, and vegetative mass of around 6%.

1. Introduction

Even though there has been some progress in ending malnutrition in the world, according to the last report of the Food and Agriculture Organization [1], between 713 and 757 million people all over the world still faced hunger in 2023 (one in 11). Moreover, according to FAO, about 2.8 billion people cannot afford a healthy diet, especially in low-income countries. Thus, there is a significant need to tackle undernutrition and micronutrient deficiencies, the so-called “hidden hunger” [2,3]. Globally, there is a widespread deficit in iron, zinc, and vitamin A [4].
As essential micronutrients, zinc (Zn) and selenium (Se) play an important role in a healthy diet and strategies against undernutrition, but their daily intake is often under adequate levels. Both micronutrients are naturally present in some foods or they can be added by various supplements in everyday dietary. Recommended dietary allowances for an adult (>19 years) are between 8 and 11 mg day−1 Zn and 55 μg day−1 Se [5]. Zn is crucial for metabolism and as the catalytic activity of enzymes which has a role in most metabolic pathways [6]. For young children pregnant women and infants, Zn is especially severe for healthy growth. The Zn deficiency leads to anomie, alopecia, delayed growth, loss of appetite, and reproductive problems [7]. In the human body, Se is important for the reproductive system, DNA formation, and general body health. The lack of Se in the human body can lead to Keshan disease (a type of heart disease), Kashin–Beck disease (a type of arthritis), male infertility, swelling, and the loss of motion in joints, but also weakens the immune system [8].
Plants are one of the most important sources of nutrition for humans and animals. A potentially sustainable and cost-effective way to alleviate the hidden hunger worldwide is the agronomic biofortification of micronutrients and their bioavailability in edible plant parts [8,9,10,11]. Usually, biofortification includes determining the dose and timing of the foliar application of Zn, Fe, Se, and I [12]. The biofortification interaction of Se and Zn has been the focus of many researchers recently [4,13]. Except for the biofortification in the later growing stages, seed priming with Se and Zn recently showed different accumulations of these elements into the plant tissue. Rice seed priming with Se (0, 30, and 60 μmol/L) increases the emergence index and total germination, as well as shoot and root lengths and dry weights [14]. Soaking seed, as a sowing pre-treatment, also leads to improving seed germination and a better establishment of seedlings [15].
The usual content of Zn in soils is 6–28 mg kg−1 [16], while Se in the soil is in the range of 0.1–2 mg kg−1 [17]. In Croatia, Se content in the soil is low and it varies from 0.024 to 9.45 mg Se kg−1 soil [13]. Plants receive selenium from the soil, water, or atmosphere through the roots and leaf surfaces. In the nature, Se can be found in inorganic and organic forms. The Se occurs as selenate (SeO42−), selenite (SeO32−), selenide (Se2−), and elemental Se, whereas in organic form Se is present as SeCys and SeMet [18]. Elemental Se, organic selenides, or metal selenides must first undergo microbial or chemical transformation to selenates before they become available to plants [19]. Selenate oxyanions (SeO42−) are the most prevalent form in agricultural soils and they are more dissolved in the soil and therefore, more available than selenite oxyanions (SeO32−). The ability to accumulate selenium in plant tissues varies. In plant tissue, SeO32− is transported by a phosphate transport mechanism, whereas SeO42− is found to be transported through sulfate transporters and channels [18]. Some plant species using sulfur (S) transporters and biochemical pathways can accumulate selenium instead of sulfur to such an extent that 0.1–1.5% of the dry weight of the plant forms Se, which does not have a toxic effect on plants [20,21]. In plants, Se can alleviate oxidative stress in the chloroplasts. Most of the reports from the literature confirmed no effect of Se on seed yield [22]. For foodstuffs, selenate or selenite can be expected to have a similar impact on the selenium’s nutritional status [23].
Zinc is contained in the soil in various forms, and its total content is quite different and depends mainly on the zinc content in the parent rock and the nature of the soil-forming process. In the soil system, Zn accumulates mainly in the humus horizon, in amounts depending on soil properties such as pH, colloid content, etc.
Zinc enters plants via the root absorption of Zn2+ from soil solution through passive nutrient transport—mass flow, root interception mechanisms (i.e., root growth), and diffusion [24]. At high pH of the soil, Zn is absorbed as ZnOH+ cation. The mobility of zinc in the plant is low. Zinc is an essential element for plants, as it is a structural element and cofactor of a number of enzymes and proteins involved in a number of biochemical processes [25]. Zinc deficiency in plants causes the inhibition of photosynthetic activity chlorosis in young leaves, shortened internodes, and decreased dry matter [26].
The usability of micronutrients highly depends on the pH of the soil solution [27,28], and the content of water, soil moisture, temperature, oxygen, iron oxide, and aluminum in the soil. In a slightly alkaline aerobic environment, the availability of selenium for plants is the highest. In calcareous soil, the availability of Zn is in deficit [29]. The availability of micronutrients generally decreases in acidic media [24].
Even though soybeans originate from China, which is widespread, nowadays, soybean is grown in over 90 countries worldwide; the biggest producers of soybeans in the world are “the big three”: Brazil, SAD, and Argentina [30]. Compared to other annual field crops, soybean is the largest protein producer per unit area [31,32,33,34,35,36]. Depending on the genotype, the amount of protein in soybeans varies from 35 to 45% [37,38], and it is widely directly or indirectly consumed. Soybean is one of the main foodstuffs for people who practice a vegetarian or vegan diet. Soybean is also used as a concentrated source of valuable nutrients in underdeveloped and developing countries where the rate of malnutrition is high. Another important use of soybeans is for livestock, which is even more important to raise quality and micronutrient status [39]. In intensive livestock production, the most important is the quality of feed, which can be highly improved by adding plants rich in micronutrients [40,41,42,43,44,45]. Soybeans as the high-protein crop are one of the best candidates for agronomic biofortification.
In a comparison of several Fabaceae such as soybean, bean (Phaseolus vulgaris L.), pea (Pisum sativum L.), lentil (Lens culinaris Med.), and chickpea (Cicer arietinum L.), Angelova et al. [46] found that soybean has the greatest tendency to absorb heavy metals from the soil with the highest accumulation in the root, then in the leaves, seeds, and least of all, in the stem. The Fabaceae family is known as hyperaccumulators of Se. Plant genotypes can vary widely in their ability to accumulate plant-available nutrients, as well as Zn and Se [24,47]. Following the above, soybeans have a special interest among scientists in biofortification due to their wide use, but also due to their geographical distribution over the world [48]. Except for soybean micronutrient biofortification, soybean biofortification with folate (vitamin B9) is also important, especially in pregnant and lactating women [49]. Kobraee et al. [50] stated that Zn concentration increased in both the roots and shoots of soybeans with increasing application rates (0 to 8 mg Zn kg−1). Moreira et al. [51] found that the soil type as one of the main factors of Zn uptake. The authors found that for BRS 133 (conventional variety), 12 kg Zn ha−1 in Typic Quartzipsamment and 6.1 kg Zn ha−1 in Orthic Ferralsol increased soybean yield, and for BRS 245RR (GM variety), the amount of 5.9 kg Zn ha−1 in Typic Quartzipsamment and 4.2 kg Zn ha−1 in Orthic Ferralsol. On the other hand, in foliar Zn application (0, 2, 4, and 6 mg L−1), Han et al. [52] did not find a significant effect on the overall soybean yield.
In Croatia, soybeans are produced mostly by oil extraction and as feed livestock due to their source of high-value proteins, with the trend of increasing the harvested area. The whole plant is often used in fresh or dried forms as animal feed. In attempts to develop Se- and Zn-enriched soybeans, the present study had the purpose of evaluating the agronomic biofortification treatment of Croatian soybean genotypes. The concentration range between the benefits and toxic effects of Zn and Se for the plants is very narrow, so this study includes the different doses of Se and Zn and evaluates their accumulation in the plant above-ground organs, with special attention to macro- (P, K, Ca, and Mg) and microelement (Fe, Mn, Cu, Zn, and Se) status in the soybean grain and their removal by the crop.

2. Materials and Methods

2.1. Field Trail and Soil Properties

Field experiments with 6 different varieties of soybeans were conducted in the growing seasons of 2020 and 2021 at the test site of the Faculty of Agrobiotechnical Sciences Osijek, at the Tenja location in the Osijek-Baranja County, Croatia. Each soybean variety was subjected to 6 different treatments of foliar biofortification (3 treatments with Se, 3 treatments with Zn, and 1 treatment of the combined application of Se and Zn), which together with the control without biofortification makes 7 different treatments, with a completely random arrangement of plots in three replications. Each individual plot had a surface area of 8 m2. Given that there was a total of 126 plots (6 varieties × 7 treatments × 3 repetitions), the total experimental area was slightly more than 1000 m2.
The soil of field experiment 2020 was alkaline (pHH2O = 8.21; pHKCl = 7.38, according to ISO 10390:2005 [53]), with medium soil organic matter content (SOM = 2.55%, according to ISO 14235:1998 [54]). The soil was very highly supplied with plant-available phosphorus (387.4 mg/kg P2O5 according to AL method [55]), and well supplied with plant available potassium (250.5 mg/kg K2O according to AL method [55]). The level of total Zn was at a moderate concentration (61.24 mg/kg) and a low concentration of total Se (0.18 mg/kg) extracted by aqua regia (according to ISO 11466:1995 [56]), as well as a medium availability of Zn (1.63 mg/kg) determined by the EDTA method [57].
The similar properties of the soil were determined before the experiment in 2021, but with a slightly more alkaline reaction, and lower contents of SOM and plant-available phosphorus: pHH2O = 8.55, pHKCl = 7.70; low soil organic matter content (SOM = 1.90%); high supplied with plant-available phosphorus (263.5 mg/kg P2O5); well supplied with plant available potassium (233.4 mg/kg K2O); a moderate concentration of total Zn (47.8 mg/kg); low concentration of total Se (0.13 mg/kg) extracted by aqua regia; and a low to medium availability of Zn (1.51 mg/kg) determined by the EDTA method.

2.2. Biofortification Treatments

In the field experiment of agronomic biofortification, 6 varieties of soybeans from the breeding program of the Agricultural Institute of Osijek (Croatia) were grown (Table 1).
The forecrop of soybean was winter wheat. Sowing was carried out in both years in optimal terms (23 April 2020 and 21 April 2021). Soybean seeds were treated immediately before sowing with microbial bioagents (50 g/10 kg of seeds) containing Bradyrhizobium japonicum. During the growth, all regular agrotechnical measurements were performed in weed control. There were no pests or disease attacks.
The experiment design was set up with soybean varieties as the main plot, and the biofortification treatment as the sub-plot.
Biofortification was conducted by the foliar application of Zn (zinc sulfate) or/and Se (sodium selenate) solutions at the beginning of the soybean flowering (second half of July).
A foliar application (with a backpack accumulator sprayer) of 1.5 L of solution (water or an appropriate solution of sodium selenate or zinc sulfate plus 1.5 mL of adjuvant) was applied to each plot of 8 m2. Each treatment was represented by three randomly arranged repetitions (plots).
The following biofortification treatments in the experiment were applied:
  • Control (without Se or Zn solutions): water was applied;
  • Se_1 treatment: 10 g/ha Se;
  • Se_2 treatment: 20 g/ha Se;
  • Se_3 treatment: 30 g/ha Se;
  • Zn_1 treatment: 3 kg/ha Zn;
  • Zn_2 treatment: 6 kg/ha Zn;
  • Se_3 Zn_2 treatment: 30 g/ha Se + 6 kg ha Zn.

2.3. Plant Material Sampling and Harvest

The sampling of plant material and harvesting of experimental plots were carried out on the same day (second half of September) for all the varieties. The sample was taken so that all the plants were collected from the surface of 1 m2 in each experimental plot by carefully cutting off the stem 2 cm above the soil surface. All the above-ground parts of the plant (stem, leaves, pods, and seed) were collected together, whereby only the leaves that were still on the stem were collected, but not the leaves that had already fallen from the stem. In this way, only the amount of above-ground mass that can be collected by conventional soybean harvesting is included in the analysis. Soybean grains were harvested from these samples, and the above-ground vegetative organs (stem, leaves, and pods) were separated and dried in a large laboratory dryer at 70 °C to a constant mass.
Also, the weight of all the soybean organs was precisely weighed, and the yield of grains, above-ground organs, and vegetative and total above-ground soybean mass was calculated from these data. Yields are expressed in t/ha of dry matter.

2.4. Analysis of Plant Material

Dry samples of plant material (stems, leaves, pods, and grain) were prepared for laboratory analysis by grinding in an ultracentrifugal mill (Retsch ZM 200, Haan, Germany), which is characterized by the fact that during the grinding of samples, additional amounts of heavy metals are not transferred into the sample. The stem, leaf, pod, and grain samples were digested in a laboratory microwave oven (CEM Mars6, Charlotte, NC, USA) using a wet analytical method [58,59]. Teflon cuvettes were used for digestion, into which 500 mg of the plant sample was quantitatively transferred, and then 8 mL of 65% HNO3 and 2 mL of 30% H2O2 were added. After digestion, the solution was quantitatively transferred through filter paper into a 50 mL volumetric flask and topped up to the mark with deionized water. The concentrations of all the elements (including Zn and Se) were measured directly in the solutions using inductively coupled plasma mass spectrometry (ICP-MS, Agilent Technologies 7800 ICP-MS, Santa Clara, CA, USA). Concentrations of most elements (including Zn) are expressed in mg/kg of dry matter, and elements with lower concentrations (including Se) are expressed in µg/kg. Measurement by mass spectrometry was performed with an internal pooled plasma control and with two appropriate reference materials (Institute of Nuclear Chemistry and Technology (INCT/ICHTJ) INCT-SBF-4 and Institute for Reference Materials and Measurements BCR 129) prepared for analysis in the same way as all the other samples.

2.5. Calculations of Removal Amount of the Nutrients and Biofortification Efficiency

The total amounts of individual elements that were removed by the above-ground mass of an individual soybean organ were calculated as the product of the yield of an individual organ and the concentration of a certain element in the respective organ. The quantities removed by the above-ground vegetative mass (stem + leaves + pod) or by the total above-ground mass (vegetative mass + grains) were calculated as the sum of the removal of certain elements by the specified individual organs. The removed amounts of an individual element are expressed in g/ha (for Zn) or in mg/ha (for Se).
The efficiency of each biofortification treatment was calculated as a percentage of the total added amount of Zn (3 or 6 kg/ha) or Se (10, 20, or 30 g/ha) found in the total above-ground mass of soybean (or only soybean grains) minus the amount of Zn or Se in the control treatment.

2.6. Statistical Data Processing

To compare the differences between all the studied parameters caused by the variation in Zn and Se levels and soybean genotype, all data were subjected to an analysis of variance (ANOVA), through the GLM procedure (general linear models), using the SAS Enterprise Guide 7.1 [60]. The Bonferroni test was used as an adjustment method for comparison and the differences between the means were presented at a probability level of 5% with Fisher’s least significant difference (LSD) test.

3. Results

3.1. Macro- and Micronutrient Status in the Soybean Grain

3.1.1. The ANOVA of the Macro- and Micronutrient Status in the Soybean Grain

According to the ANOVA analysis of the macro- (P, K, Ca, and Mg) and micro- (Fe, Mn, Cu, Zn, and Se) elements (Table 2), the soybean varieties differed very significantly (p < 0.0001) for all the elements, except in the uptake of K and Cu in the grain. Biofortification treatment has a very significant influence on P, Mn, Zn, and Se content in the soybean grain.
All the treatments and their interactions had a very significant influence on Zn accumulation in the soybean grain, whereas in the case of Se, significance was not found only within the interaction of the year (Y) and variety (V).

3.1.2. Macro- and Microelement Accumulation in the Grain

On average for both years in this study, the Zn and Se biofortification treatment showed different influences on macro- and microelements content in soybean grain (Figure 1a–g).
For macronutrient status, the biofortification treatments showed significant influence only in the case of accumulated P in the soybean grain (Figure 1a–d). The highest content of P in this study was determined in the control treatment (6.89 g/kg), whereas biofortification treatments decreased the content of P (Figure 1a). In regard to varieties, the Toma variety had the highest accumulation of P (7.19 g/kg), and the Sonja variety accumulated less P (6.22 g/kg). The biofortification treatments nor variety did not have a significant influence on the average K content in the soybean grain, which was 20.06 g/kg (Figure 1b). For the Ca content, there were no significant differences among the biofortification treatments, whereas for the varieties, the differences among the mean accumulated Ca were significant (Figure 1c). The Ca content varied from 2.00 g/kg for the Ika variety to 2.82 g/kg for the Toma variety. The Mg content in the soybean grain was, on average, for all treatments, 2.54 g/kg (Figure 1d). Here, the varieties showed significant differences in relation to the accumulation of Mg, so the Sunce variety accumulated the highest Mg content (2.78 g/kg) and the Sonja variety accumulated the lowest amount of Mg (2.41 g/kg).
The Fe and Mn content in the soybean grain were significantly different among biofortification treatments (Figure 1e,f). Biofortification with Zn slightly decreased the Fe content in the soybean grain, and at the same time increased the Mn content. The Fe content on the Se biofortification treatments was on average 77.94 mg/kg, whereas with Zn foliar application, the Fe content was on average 74.58 mg/kg (Figure 1e). The lowest content of Fe in the grain was determined at the Se_3 Zn_2 treatment, which was 12.5% lower than the control. Among the varieties, the Fe content varied from 65.0 mg/kg (Lucija) to 82.3 mg/kg (Toma). Biofortification significantly influenced the Mn status in the soybean grain (Figure 1f), which was with Se foliar application on average 29.24 mg/kg and with Zn and simultaneous Se and Zn application on average 32.31 mg/kg. Soybean varieties in this study showed significant differences in Mn accumulation, which was the highest for the Lucija variety (33.07 mg/kg) and the lowest for the Ika variety (25.96 mg/kg). The Cu content in the soybean grain was on average 17.66 mg/kg, and it was not significantly affected by biofortification or variety (Figure 1g).

3.1.3. Zn and Se Accumulation in the Grain

In this study, the biofortification treatments have a very significant influence (p < 0.001) on the Zn and Se content in the soybean seeds (Table 2).
Foliar biofortification with Se did not increase Zn content, which was on average 45.92 mg/kg, and it was not significantly different from the control treatment (Figure 2a). With Zn application, the significant differences were determined between the lower (Zn_1) and higher (Zn_2) dose of applied Zn; thus, the highest accumulation was on average 66.14 mg/kg with Zn_2 and simultaneous Se_3 and Zn_2 application, which was about 3% higher compared to the Zn_1 treatment. The soybean varieties have different responses to foliar biofortification with Zn. The Toma variety has the highest content of Zn in the grain (61.47 mg/kg), which was significantly different from the other varieties. The lowest content of Zn in the grain was found for Lucija and Sonja (50.91 mg/kg on average), with no significant differences between them.
Selenium biofortification has a great impact on increased Se content in the grain (Figure 2b). Without Se foliar application (Zn_1 and Zn_2 treatment), the Se content in the grain was at the control level (55.29 µg/kg), with no significant differences determined between the control and Zn treatments. The foliar application of Se vigorously increased the Se grain content, which was increased linearly with increasing Se dose. Thus, the Se content in the grain was 1155.43 µg/kg at the Se_1 treatment, and increased by 77.5% at the Se_2 treatment and even more, by 156%, at the Se_3 treatment. Regarding the varieties, the lowest accumulation of Se in the soybean grain was in the Lucija grain (1070.71 µg/kg) and the highest amount of Se was found in the grain of the Sunce variety (1436.85 µg/kg).

3.2. Distribution of Zn and Se in Above-Ground Organs

To determine the accumulation of the Zn and Se in different above-ground organs of soybean plants, the leaves, grain, pods, and stems were separately analyzed. The biofortification treatments have different influences on Zn and Se uptake by the different plant parts (Figure 3a). The highest accumulation of Zn was determined in the leaf of Zn_2 (388.87 mg/kg), then on the treatment Se_3 Zn_2 and Zn_1, where the leaves accumulated a similar amount of Zn (333.96 and 333.15 mg/kg, respectively). The Zn biofortification treatments have a positive influence on Zn accumulation in the grain, and on average, for all the Zn treatments, the grain accumulated 65.41 mg/kg, which was 41% higher compared to the control treatment.
On the opposite of Zn accumulation, the highest accumulation of Se was in the soybean grain than in the other above-ground organs (Figure 3b). Generally, soybean biofortification increased Se content in all the plant parts as compared to the control treatment. Thus, the content of Se in the soybean grain with the Se_1 foliar application was 1155.43 µg/kg, which was about 21 times higher than the control, and even more at the Se_3 treatment (2950.18 µg/kg) where it was about 53 times higher compared to the control. The highest Se leaf content was determined on the Se_3 Zn_2 treatment (1145.45 µg/kg), which was about 20 times higher compared to the control. It was interesting to find that the stem Se content was also increased from 17.65 µg/kg (control) to 246.60 µg/kg on average for all the Se treatments.
The varieties of soybean differed in Zn and Se accumulation (Figure 4). On average of both years and biofortification treatments, for all the above-ground tissues (Figure 4a), Lucija (86.98 mg/kg) and Sonja (78.18 mg/kg) accumulated the highest rate of Zn, whereas for the other varieties, accumulation varied on average from 53.20 mg/kg (Ika) to 59.61 mg/kg (Toma). For Se (Figure 4b), Sunce and Toma showed the highest accumulation of Se in above-ground tissues (651.30 and 568.65 µg/kg, respectively). The Lucija variety accumulates the lowest content of Se in the above-ground tissues (total 453.86 µg/kg).

3.3. Linear Regression of Biofortification and Grain Zn and Se

There was a significant and positive correlation between the Zn and Se treatments and their grain content in both years of the study (Figure 5a,b). According to the simple linear regression, for each kg of increased Zn dose, the amount of Zn in the soybean grain increases by 3.18 mg/kg (Figure 5a). For the Se, the single linear regression showed a significant relationship between the foliar Se and Se grain content. Thus, it was calculated that for each g of Se increment in foliar application, the Se content in the soybean grain will increase by 338.71 µg/kg (Figure 5b).

3.4. Macro- and Micronutrient Removal by the Soybean

3.4.1. The ANOVA of the Macro- and Micronutrient Rem Oval by the Plant

The ANOVA analysis was performed to determine the effect of all parameters on soybean grain and vegetative mass yield and the removal of the macro- and micronutrients with the plant (Table 3).
In general, all the treatments and their interactions have a significant influence on the Zn and Se removal by the plant. Moreover, for all the macro- (P, K, Ca, and Mg) and microelements (Fe, Mn, Cu, Zn, and Se), there were significant differences determined among the soybean varieties.

3.4.2. Macro- and Micronutrient Removal by the Plant

Since the varieties showed very significant differences (p < 0.001) in the removal of all the analyzed micro- and macronutrients (Figure 6a–i), the removal elements by the grain and vegetative mass were described in detail. The vegetative mass represents all above-ground tissues except the grain. Phosphorus removal by the plant was higher with the grain (25.38 kg/ha), while the vegetative soybean mass P removal was on average 3.20 kg/ha (Figure 6a). The Toma variety had the larger P removal by the crop. In the case of K, the higher content of removed K was determined for the grain, and the lowest for the other above-ground tissues. The Toma variety has the highest K removal in grain and vegetative mass (Figure 6b). The Ca removal by the grain was less accelerated, while for vegetative mass, the Toma variety’s removal of Ca was the highest.
The average Mn and Fe removal was higher for plants with vegetative mass than for grain removal (Figure 6d,e). The Ika, Korana, and Toma varieties stand out in the Mg and Fe removal by the grain (Figure 6d,e). For Mg removal (Figure 6d), the varieties Ika and Toma stand out for their removal of Mg in vegetative mass, whereas in the case of Fe, Toma and Korana varieties removed the highest amount of Fe in the vegetative mass. The Toma variety removes the highest amount of Mn in all plant above-ground tissues (Figure 6f). The varieties Ika and Toma are the highest in Cu removal by the grain (Figure 6f), and the Toma variety removes the highest dose of Cu in the vegetative mass. For the Zn and Se removal by the variety, the Toma variety had the highest removal of both micronutrients added by the biofortification in the grain (Figure 6h,i). Except for the Toma variety, the varieties Korana and Ika stand out for Zn and Se removal in the soybean grain (Figure 6h). The Lucija and Sunce varieties stand out for Zn removal by the vegetative mass (Figure 6h), whereas Sunce and Toma had the highest Se removal in the vegetative mass (Figure 6i).

3.5. Nutrient Use Efficiency of Zn and Se Treatments

The nutrient use efficiency (NUE) of Zn and Se biofortification treatments in soybean crops was calculated (Table 4). The biofortification treatments did not influence grain yield nor vegetative mass yield (all above-ground organs except the grain). This study’s average soybean vegetative mass and grain yield were similar (3.9 t/ha).
The NUE of Se was the highest with the application of 10 g/ha Se (Se_1) and 20 g/ha Se (Se_2) where it was on average 43.2% for the grain and 6.7% for vegetative mass (stem, pods, and leaves). The Zn NUE was the highest with 3 kg/ha foliar treatment for both grain and vegetative mass (2.6% and 4.4%, respectively), which was significantly different (p < 0.05) from the other Zn treatments.

3.6. Categorization of Soybean Varieties for NUE

The Zn NUE of soybean grain was the highest for the Korana variety (2.68%) and the lowest for the Ika variety (1.19%) (Figure 7).
For Se grain, NUE was the highest in the Sunce variety (29.15%) and the lowest in the Ika variety (20.15%). Within the soybean genotypes, the NUE of the vegetative mass was the highest for Zn (Figure 7) in the Lucija and Toma varieties (4.03%), whereas the lowest Zn NUE was found in the Ika variety (2.18%). The Sunce, Toma, and Ika varieties had the highest Se NUE for vegetative mass (7.1% on average) and the Sonja, Lucija, and Korana varieties had lower (4.3% on average).

4. Discussion

This study gives a comprehensive view of Zn and Se biofortification in different soybean genotypes. The accumulation of macro- and microelements differs among the biofortification treatments and soybean varieties, as well as their removal by the plant.
Nutrient removal with harvest is an important factor of sustainability nutrient management strategies of the production systems [61,62,63,64]. In this study, the differences in nutrient status in the soybean grain were more expressed among varieties than among biofortification treatments (Figure 1). Esper Neto et al. [65] found that newer Brazilian soybean genotypes increased nutrient removal with 11.1, 26.9, 45.0, and 31.6% for N, P, K, and Mg, respectively, removed compared to those grown in the past. Moreover, the authors stated that P showed the removal of 4.95 kg/t, and K of 20.6 kg/t of soybean grain yield. The breeding programs were more focused on maximizing the grain yield [66,67], but nowadays, the genetic approach and transfer of the nutrients into the plant tissue should also be taken into consideration, which requires a new research approach [68]. Except for the genotype, Lončarić et al. [55] stated that various factors, such as soil temperature, pH, microbiological activity, and moisture, influence the accumulation of the elements in plant organs. Even though plants can absorb large amounts of some nutrients, this may not increase their yield, but it can have a huge influence on the yield quality [69,70,71]. Nutrient removal is very important to adjust plant’s needs for the nutrients added by the fertilizer and increase NUE. For three peanut (Leguminosae family) varieties, Crusciol et al. [72] found the average nutrient removal by the pods followed the order P > N > K > S > Mg > Ca. With the meta-data analysis, Filippi et al. [73] showed differences in soybean grain micronutrient status over the years (1974–2019) in Brazil. The authors stated that the N content of the soybean grain increased by 1.1 kg for each tonne of seed yield and that Ca, Mg, and S were not affected by the seed yield. In the present study, the biofortification treatment decreases the content of P in the soybean seed (Figure 1a). Santos et al. [74] reported that the physiological response of the P × Zn interaction in plants remains poorly understood. Although both P and Zn are essential elements for plants, in certain circumstances, they can act antagonistically to each other, which can lead to a decrease in yield, either due to P or Zn deficiency [75]. Antagonism most often occurs when one of the elements is present in large quantities. Most often, Zn deficiency in plant tissues occurs with excessive P application, but P deficiency with excessive Zn application is also possible, although rarely. Santos et al. [74] reported that for cotton plants, excessive P uptake reduces Zn shoot concentration and that Zn supply regulated acid phosphatase activity which facilitated adaptation responses to P deficiency.
In this study, the biofortification Zn treatment significantly increases Mn content in the soybean grain (Figure 1a). Manganese has a favorable effect on the formation of nodules in soybeans and therefore, it is an important nutrient for soybeans. In the plants, Mn and Zn interact with each other, which can have a possible impact on the yield [75,76,77]. In sweet corn (Zea mays var. saccharata), Soltangheisi et al. [78] found Mn and Zn in the way that Zn content in the roots increases with increasing Mn levels, whereas increasing levels of Zn in the nutrient solution decreased Mn concentration in roots. Dhaliwal et al. [79] reported that Zn and Mn foliar application helped with the absorption of N, P, and K.
Higher concentrations of Zn and Se in the grain and vegetative mass confirm the effectiveness of the biofortification treatments. However, some of the genotypes were found to be superior to others. In the present study, the interaction of biofortification and variety (Table 1) for Zn content was very significant (p < 0.0001). The Toma variety accumulated the highest amount of Zn in the Se_3 Zn_2 treatment (76.80 mg/kg), whereas the lowest Zn content was in the Korana variety in the Se_2 treatment (41.49 mg/kg). The interaction of biofortification and variety (Table 1) in Se content in the soybean grain was very significant (p < 0.0001). The Ika variety at the Zn_2 treatment had the lowest Se content (26.77 µg/kg), whereas the highest Se content was found for the Sunce variety at the Se_3 treatment (3601.37 µg/kg). It was interesting to find that the highest increment of Se content, compared to the control, was in the Ika variety, where Se content increased about 75 times at the Se_3 treatment. The interactions of variety and biofortification showed the lowest increment of Se in the grain in comparison to the control for the Lucija and Toma varieties, whose increment was about 49 times compared to the control at the Se_3 treatment. Moreover, within the categorization of soybean genotypes over two years for the NUE of the soybean grain, the Korana variety had the highest NUE for Zn and Toma for Se. For vegetative mass, the highest NUE of Zn and Se were found for the Toma variety. These findings can be very valuable for the efficiency and sustainability of agricultural systems to increase NUE [80]. Galić et al. [81] state that the cultivars Sonja and Lucija have different physiological responses to Se biofortification, where a higher dose of Se in biofortification decreases proline content in the Lucija variety and on the other hand effect negatively in the Sonja variety, where it is possible that it could act as pro-oxidant. The consumption of foods containing less than 0.1 mg Se kg−1 results in selenium deficiency in the body, while the regular consumption of foods containing more than 1 mg Se kg−1 is toxic [14].
Low concentrations of Zn and Se in the grain are mostly related to their low content in the soil system. In the present study, foliar biofortification greatly improved Zn and Se content in the soybean grain. According to Sadeghzadeh [24], Zn-efficient plant genotypes have the ability to absorb higher doses of Zn may contain a higher dose of chelators, which bind the Zn and increase its physiological availability in the cells. Rose et al. [82] investigated the effect of foliar fertilization with zinc (ZnSO4·7H2O) on yield increase in four soybean cultivars (Lee, Forrest, Bragg, and Dodds) in New South Wales. Foliar Zn application in the stage before flowering led to an increase in yield in all four genotypes, the plants were taller, had a higher content of oil in the grain (in two varieties), and the zinc content in the leaves was increased and the phosphorus content was reduced. Dai et al. [83] stated that the biofortification of Se and Zn in soybeans was different among the elements in that the application of Se alone reduced Zn uptake. In contrast, the application of Zn promoted both Se and Zn in grains. Silva et al. [84] reported that Se content in soybean grain was about three times greater with an application of MAP (~50% P2O5) + Se (rate of 80 g ha−1) fertilizer compared to phosphorus fertilizers without Se. In wheat (Triticum aestivum L.), Kong et al. [85] also found a huge increment in Zn and Se with biofortification, Zn by 12.07–71.88% (up to 41.66–64.30 mg kg−1), and grain Se content by 131.81–527.21% (up to 0.21–0.50 mg kg−1).
Hossain et al. [86] reported that in the production of Se-enriched food, a wider approach is needed through agronomic Se biofortification combined with breeding and molecular and biotechnology. The production of Se- and Zn-enriched soybean is especially important because soybean is one of the main and valued protein nutrients, not only for seed and flour but also for straw and dried green material [87,88,89,90]. In the present study, a detailed Zn and Se biofortification gives a new perspective on the production of Zn- and Se-enriched soybeans, which is valuable not only for the production of Zn- and Se-enriched grain but also in the production of vegetative mass of soybean which is used as nutritive enriched animal feed. Therefore, biofortified varieties should be superior to the less nutrient varieties and adopted as prioritized products of family farming.

5. Conclusions

The biofortification strategy represents one of the most important measures to improve the quality of the grain and/or other edible plant parts. For successful biofortification of crops, it is important to determine the dose for foliar application and consider the dose that is desired in the edible plant part. Soybean is a highly nutritional crop; thus, it is very interesting among researchers for biofortification. The present study found that six soybean genotypes are very variable in the nutrient’s uptake and removal, as well as their NUE for Se and Zn biofortification treatment. Biofortification treatment did not show a significant influence on soybean grain or vegetative mass yield, but evident differences were found in Se and Zn accumulation in the soybean grain among treatments and varieties. This study has made a great contribution to so-called “next-generation” cropping systems, which focus on improving nutrient use efficiency in relation to raising soil health and minimizing the use and application of mineral fertilizers.

Author Contributions

Conceptualization, Z.L., A.S., V.Z. and F.N.; methodology, Z.L., L.G. and A.S.; software, I.V.; validation, Z.L. and A.S.; formal analysis, I.V., F.N., K.P., J.J., V.Z., V.I., D.I. and L.G.; investigation, K.P., J.J., V.Z., V.I., D.I. and L.G.; resources, A.S.; data curation, Z.L., V.I. and J.J.; writing—original draft preparation, Z.L. and I.V.; writing—review and editing, Z.L. and A.S.; visualization, I.V.; supervision, Z.L.; project administration, Z.L. and D.I.; funding acquisition, Z.L. All authors have read and agreed to the published version of the manuscript.

Funding

The paper is a result of the research conducted within the project KK.01.1.1.04.0052 “Innovative production of organic fertilizers and substrates for growing seedlings” funded by the European Union in the framework of the 2014–2020 Competitiveness and Cohesion Operational Program of the European Regional Development Fund.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The raw data supporting the conclusions of this article will be made available by the authors upon request.

Conflicts of Interest

The authors declare no conflicts of interest.

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Applsci 14 11349 g001
Figure 1. The average macro- and micronutrients in the soybean grain in relation to the biofortification treatment and variety, (a) P (g/kg), (b) K (g/kg), (c) Ca (g/kg), (d) Mg (g/kg), (e) Fe (mg/kg), (f) Mn (mg/kg), (g) Cu (mg/kg) (means denoted by a different letter indicate significant differences between the biofortification treatments and between varieties at p < 0.05).
Figure 1. The average macro- and micronutrients in the soybean grain in relation to the biofortification treatment and variety, (a) P (g/kg), (b) K (g/kg), (c) Ca (g/kg), (d) Mg (g/kg), (e) Fe (mg/kg), (f) Mn (mg/kg), (g) Cu (mg/kg) (means denoted by a different letter indicate significant differences between the biofortification treatments and between varieties at p < 0.05).
Applsci 14 11349 g001
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Figure 2. The average (a) Zn and (b) Se status in the soybean grain in relation to the biofortification treatment and variety (means denoted by a different letter indicate significant differences between the treatments of biofortification and varieties at p < 0.05).
Figure 2. The average (a) Zn and (b) Se status in the soybean grain in relation to the biofortification treatment and variety (means denoted by a different letter indicate significant differences between the treatments of biofortification and varieties at p < 0.05).
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Figure 3. Treemap diagram of Zn (a) and Se (b) of different above-ground plant parts (L—leaf, G—grain, P—pod, and S—stem) in relation to biofortification treatments.
Figure 3. Treemap diagram of Zn (a) and Se (b) of different above-ground plant parts (L—leaf, G—grain, P—pod, and S—stem) in relation to biofortification treatments.
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Figure 4. Treemap diagram of Zn (a) and Se (b) as the average of all the plant parts (leaf, grain, pod, and stem) in relation to the soybean variety.
Figure 4. Treemap diagram of Zn (a) and Se (b) as the average of all the plant parts (leaf, grain, pod, and stem) in relation to the soybean variety.
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Figure 5. Relationship between Zn or Se foliar application rates and Zn (a) or Se (b) content in soybean grain (blue marks represent 2020 and red 2021 year). The significance is marked as p ≤ 0.01 **.
Figure 5. Relationship between Zn or Se foliar application rates and Zn (a) or Se (b) content in soybean grain (blue marks represent 2020 and red 2021 year). The significance is marked as p ≤ 0.01 **.
Applsci 14 11349 g005
Applsci 14 11349 g006aApplsci 14 11349 g006b
Figure 6. Removal amount of macro- and microelements with grain and vegetative mass in soybean varieties: (a) P (kg/ha), (b) K (kg/ha), (c) Ca (kg/ha), (d) Mg (kg/ha), (e) Fe (g/ha), (f) Mn (g/ha), (g) Cu (g/ha), (h) Zn (g/ha), (i) Se (mg/ha).
Figure 6. Removal amount of macro- and microelements with grain and vegetative mass in soybean varieties: (a) P (kg/ha), (b) K (kg/ha), (c) Ca (kg/ha), (d) Mg (kg/ha), (e) Fe (g/ha), (f) Mn (g/ha), (g) Cu (g/ha), (h) Zn (g/ha), (i) Se (mg/ha).
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Figure 7. The NUE of Se and Zn within the soybean varieties (means denoted by a different letter indicate significant differences between biofortification treatments at p < 0.05).
Figure 7. The NUE of Se and Zn within the soybean varieties (means denoted by a different letter indicate significant differences between biofortification treatments at p < 0.05).
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Table 1. Soybean varieties of the experiment.
Table 1. Soybean varieties of the experiment.
VarietyMaturity GroupSowing Rate (Plants/ha)
Ika0–I560.000
Korana00620.000
Lucija00–0600.000
Sonja0580.000
Sunce0–I520.000
Toma0610.000
Table 2. Analysis of variance (ANOVA) for the year (Y), soybean variety (V), and Zn and Se biofortification treatments (B) for macro- and micronutrients in the soybean grain.
Table 2. Analysis of variance (ANOVA) for the year (Y), soybean variety (V), and Zn and Se biofortification treatments (B) for macro- and micronutrients in the soybean grain.
Source of
Variation		PKCa MgFeMnCuZnSe
mg kg−1μ kg−1
Y<0.0001<0.0001<0.0001ns<0.0001<0.0001<0.0001<0.0001<0.0001
V<0.0001ns<0.0001<0.0001<0.0001<0.0001ns<0.0001<0.0001
B<0.0001nsnsns*<0.0001ns<0.0001<0.0001
Y × V<0.0001<0.0001<0.0001***<0.0001<0.0001<0.0001ns
V × B nsns<0.0001nsns<0.0001ns<0.0001<0.0001
Y × B*nsnsnsns<0.0001**<0.0001<0.0001
Y × V × Bnsnsnsnsns<0.0001**<0.0001<0.0001
ns: non-significant; * significant at p < 0.05; ** significant at p < 0.01.
Table 3. Analysis of variance (ANOVA) for the year (Y), soybean variety (V), Zn and Se biofortification treatments (B), and grain and vegetative mass (P) yield with macro- and micronutrient removal by the plant.
Table 3. Analysis of variance (ANOVA) for the year (Y), soybean variety (V), Zn and Se biofortification treatments (B), and grain and vegetative mass (P) yield with macro- and micronutrient removal by the plant.
Source of
Variation		Plant Part YieldRemoval
PKCa MgFeMnCuZnSe
kg ha−1g ha−1mg ha−1
Y*<0.0001<0.0001ns**<0.0001<0.0001ns<0.0001<0.0001
V<0.0001<0.0001<0.0001<0.0001<0.0001<0.0001<0.0001<0.0001<0.0001<0.0001
Bnsnsns**nsns<0.0001ns<0.0001<0.0001
Pns<0.0001<0.0001<0.0001<0.0001<0.0001**<0.0001<0.0001<0.0001
Y × V<0.0001<0.0001<0.0001<0.0001<0.0001**<0.0001<0.0001<0.0001<0.0001
Y × Bnsnsns**nsns***<0.0001<0.0001
Y × Pns**<0.0001*ns<0.0001<0.0001<0.0001**<0.0001
V × Bnsnsns<0.0001nsnsnsns<0.0001<0.0001
V × P<0.0001<0.0001ns<0.0001**ns<0.0001<0.0001<0.0001<0.0001
B × Pnsnsns**nsnsnsns<0.0001<0.0001
Y × V × B**ns**<0.0001**ns****<0.0001<0.0001
Y × V × P**<0.0001**<0.0001*******<0.0001<0.0001
Y × B × Pnsnsns*nsnsnsns<0.0001<0.0001
V × B × Pnsnsns<0.0001nsnsnsns*<0.0001
Y × V × B × Pnsnsns<0.0001nsnsnsnsns<0.0001
ns: non-significant; * significant at p < 0.05; ** significant at p < 0.01.
Table 4. Zn and Se biofortification NUE in soybean.
Table 4. Zn and Se biofortification NUE in soybean.
Applied Foliar Fertilizer Yield
(t/ha)		Zn Removal
(g/ha)		Se Removal
(mg/ha)		Zn Use Efficiency
(%)		Se Use Efficiency
(%)
Grain
Control3.9180.1 b212.0 e--
Se_1 (10 g/ha Se)4.0253.0 b4527.1 d-43.2 a
Se_2 (20 g/ha Se)4.0184.7 b8136.3 c-39.6 a
Se_3 (30 g/ha Se)3.9176.7 a11391.1 a-37.3 ab
Zn_1 (3 kg/ha Zn)4.0257.7 a200.8 e2.6 a-
Zn_2 (6 kg/ha Zn)3.9259.3 a165.0 e1.3 b-
Se_3 Zn_2 (30 g/ha Se + 6 kg ha Zn)3.8184.0 a9700.3 b1.2 b31.6 c
Average 3.9213.64904.72.637.9
Vegetative mass
Control3.723.4 c77.0 e--
Se_1 (10 g/ha Se)4.024.4 c745.1 d-6.7 a
Se_2 (20 g/ha Se)4.024.4 c1240.0 c-5.8 ab
Se_3 (30 g/ha Se)3.825.6 c1788.2 a-5.7 ab
Zn_1 (3 kg/ha Zn)4.0156.8 b146.5 d4.4 a-
Zn_2 (6 kg/ha Zn)3.9187.14 a81.0 d2.7 b-
Se_3 Zn_2 (30 g/ha Se + 6 kg ha Zn)3.8196.8 a1472.3 b2.9 b4.7 b
Average 3.991.2792.93.65.7
Means denoted by a different letter indicate significant differences between biofortification treatments at p < 0.05.
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MDPI and ACS Style

Lončarić, Z.; Varga, I.; Nemet, F.; Perić, K.; Jović, J.; Zebec, V.; Ivezić, V.; Iljkić, D.; Galić, L.; Sudarić, A. Efficiency of Biofortification with Zn and Se in Soybean: Yield and Overall Mineral Content in Plant. Appl. Sci. 2024, 14, 11349. https://doi.org/10.3390/app142311349

AMA Style

Lončarić Z, Varga I, Nemet F, Perić K, Jović J, Zebec V, Ivezić V, Iljkić D, Galić L, Sudarić A. Efficiency of Biofortification with Zn and Se in Soybean: Yield and Overall Mineral Content in Plant. Applied Sciences. 2024; 14(23):11349. https://doi.org/10.3390/app142311349

Chicago/Turabian Style

Lončarić, Zdenko, Ivana Varga, Franjo Nemet, Katarina Perić, Jurica Jović, Vladimir Zebec, Vladimir Ivezić, Dario Iljkić, Lucija Galić, and Aleksandra Sudarić. 2024. "Efficiency of Biofortification with Zn and Se in Soybean: Yield and Overall Mineral Content in Plant" Applied Sciences 14, no. 23: 11349. https://doi.org/10.3390/app142311349

APA Style

Lončarić, Z., Varga, I., Nemet, F., Perić, K., Jović, J., Zebec, V., Ivezić, V., Iljkić, D., Galić, L., & Sudarić, A. (2024). Efficiency of Biofortification with Zn and Se in Soybean: Yield and Overall Mineral Content in Plant. Applied Sciences, 14(23), 11349. https://doi.org/10.3390/app142311349

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