Seedling Priming with Selenium Enhances the Biofortification Strategies in the Production of Broccoli Florets

Abstract

Agronomic biofortification strategies have been used to increase selenium (Se) concentrations in edible parts, with broccoli cultivation showing high potential. Recent studies have demonstrated that prior application of selected elements during the seedling phase (priming) can enhance agronomic biofortification when this element is applied during the adult phase; however, no such effect has yet been reported for Se. Additionally, Se concentration in broccoli florets may be affected by post-harvest processing, thus determining losses is essential in the agronomic biofortification process. This study aimed to determine whether seedling production with priming using selenium (Se) could enhance different agronomic biofortification strategies for Se, and to evaluate the effect of post-processing on the Se concentration in broccoli. Seedlings were produced with and without priming (75 mg L⁻¹ of Se), and different application methods (soil and foliar), sources, and doses of Se were tested on Se concentration in broccoli florets. Foliar application strategies for Se were more effective than soil application for producing Se-biofortified broccoli. Seedlings produced and subjected to Se application to promote the priming effect enhanced Se absorption and increased Se concentration in broccoli florets. However, the highest Se absorption with a dry mass concentration exceeding 18 mg kg⁻¹ reduced broccoli production, except for Se applied via multi-nutrient fertilizer. Foliar fertilization strategies using 50 g of Se ha⁻¹ via multi-nutrient fertilizer, Se + organic compounds, and sodium selenate, along with the use of seedlings produced with priming and the application of 50 g of Se ha⁻¹ via multi-nutrient fertilizer using seedlings produced without priming, can provide Se amounts reaching the human dietary requirement of 60–70 µg day⁻¹, based on the adequate daily consumption of broccoli (40 g of broccoli). Different processing stages do not cause significant losses of Se in biofortified florets. Therefore, it is concluded that seedlings produced with priming combined with foliar Se applications are effective strategies for promoting agronomic biofortification of Se in broccoli florets for the human diet.

1. Introduction

Selenium (Se) is involved in various physiological and metabolic processes, including the immune response and antioxidant defense system [1,2,3,4]. The intake of Se in the human diet is generally low, primarily due to the low natural availability of Se in the soil, which consequently limits its accumulation in edible plant organs [5,6,7]. In addition to low Se intake, the bioavailability of Se and its beneficial effects against diseases are highly dependent on the chemical form in which Se is present [4,8,9]. Organic forms derived from plant products are more efficient in preventing certain diseases than inorganic Se forms [8,10]. Moreover, Se in organic forms is more bioavailable and less toxic [9,11].

The primary source of Se for humans is plants, as they can metabolize and accumulate organic forms of this element in parts that can be directly ingested (e.g., leaves, flowers, fruits, seeds, and sprouts) or indirectly after processing (e.g., oil and wine). Recently, it has been demonstrated that plant biofortification can produce more nutritious foods, benefiting human health when these biofortified foods are incorporated into the diet [6,7,12,13].

Among the plants that can be biofortified and are staples in the human diet, those of the Brassica L. genus, such as broccoli, are naturally efficient at accumulating Se [5,6,13,14,15]. Additionally, broccoli is rich in vitamins, dietary fiber, and minerals, which are sources of anti-carcinogenic phytochemicals, such as polyphenols and sulfur- and nitrogen-rich compounds [13,16,17]. The potential of broccoli for Se biofortification is attributed to its high level of sulfur compounds. Selenium can partially replace sulfur in certain compounds, such as in the formation of selenoproteins [1,6,14,15]. These properties of broccoli are excellent for promoting biofortification with Se. However, the nutritional quality of broccoli and its Se concentration is highly dependent on storage and processing conditions, such as blanching and freezing [18,19,20]. Therefore, it is necessary to study the nutritional quality of Se biofortified foods after processing.

Among the strategies to increase the concentration of human-essential elements in edible parts, genetic and agronomic biofortification stand out, with the latter being more effective for Se [6,7]. Agronomic biofortification involves applying a specific target element through fertilization processes to promote greater accumulation in the edible part of the plant, thereby allowing for an increase in the intake of this particular element by humans after consumption [6,12]. Several studies in different countries, including Brazil, corroborate the efficiency and environmental safety of this nutritional enrichment method. Various crops and target elements are used for this purpose, with Se being one of the elements with high potential for biofortification purposes [12,21,22]. However, scientific studies on effective strategies for broccoli biofortification with Se are still uncommon in Brazil. In addition, the results show the potential of this crop for inclusion in the agronomic biofortification process with Se [10,13,14,15,23].

Agronomic biofortification relies on various interacting factors, including application method, dose, application timing, source applied, soil type, and plant species [1,5,6,12,22,24]. Therefore, despite being a promising technique, it is necessary to test different strategies in commercial production systems to guarantee the effectiveness of the biofortification process. These strategies must ensure the maintenance or even the increase in production, as well as the enhancement of the nutritional quality of the produced food [12]. The interaction of these factors provides different mechanisms for the absorption, translocation, and efficiency of the applied elements [6,24,25]. These factors are highly dependent on plant species and, within the same species, the genotype to be biofortified with Se [6,24]. In addition, Se application affects plant gene expression, potentially triggering and promoting specific metabolic processes that may stimulate Se absorption and/or induce resistance to adverse stress conditions [26,27].

A promising yet underexplored technique that may enhance the stimulation of carriers involved in Se absorption is the prior application of Se through the application of solutions containing the target element for biofortification on seedlings or seeds. This process can stimulate factors that may eventually improve the performance and absorption of the target element (Se) in the biofortified product, a phenomenon known as the priming effect [11,26,28,29,30,31]. The priming effect is related to the potential stimulation of Se during prior application on processes that trigger various chemical reactions, such as stimulation of Se carriers, metabolic activity, and production of chemical compounds. Thus, the priming effect is defined as the pre-exposure of plants to a specific factor that can trigger responses related to “metabolic memory”, allowing plants to develop better under subsequent stress conditions [26,32,33].

The priming effect can enhance both the capacity for Se accumulation in subsequent applications at different growth stages and the initial development of plants or even create metabolic mechanisms that ensure greater resilience in adverse field conditions [26,32,33,34]. As a result, increased functionality of this metabolic mechanism is expected, which may consequently improve Se absorption by plants. The priming effect of Se application on rice seeds, depending on Se concentration applied, promotes faster and more remarkable seed emergence and stimulates the plant’s metabolic system, directly influencing Se concentration in polished grains under field conditions [34]. Some studies with broccoli show the potential to improve the plant’s development and its antioxidant defenses. Still, there are no studies that show how this can maximize the agronomic biofortification process [26,35].

Another critical aspect of biofortification is the availability of Se sources currently on the market. Several available products may contain other chemical compounds and elements, which may or may not enhance Se absorption by plants and/or promote simultaneous biofortification with other elements, mainly when Se is applied through multi-nutrient fertilizer containing Se (SeMNF) [12,36].

In this context, this study hypothesizes that seedlings with a Se priming effect can maximize the agronomic biofortification process with Se in broccoli florets, with foliar application being more effective than soil application. The study objectives were: (i) to evaluate how the addition of Se during the seedling production phase induces a priming effect when this elements is later applied in broccoli plants during the inflorescence production phase (florets); (ii) to assess how supplementary Se addition improves the nutritional quality of broccoli, while also positively impacting agronomic characteristics of the crop; (iii) to evaluate how the biofortification effectiveness varies according to Se doses and sources (organic vs. inorganic), application methods (soil vs. foliar), and application stages (seedling vs. vegetative vs. reproductive); (iv) to determine how different post-harvest processing strategies affect Se concentration in biofortified broccoli florets.

2. Materials and Methods

2.1. Treatments and Experimental Conditions

Broccoli seedlings (Brassica oleracea var. italica) of the cultivar Avenger were produced with and without priming effect. The seedlings were grown in polystyrene trays containing a nutrient solution [37]. These seedlings were transferred to trays with commercial substrate until they reached the optimal transplant stage (45 days after planting, when seedlings had 4–6 leaves per plant) under conditions similar to those used in commercial field cultivation. In the priming seedlings, 7 days before transplanting, a foliar application of Se was applied at a concentration of 75 mg L⁻¹ in the form of sodium selenate (Na₂SeO₄) (reagent grade, Sigma Aldrich, St. Louis, MI, USA), along with 0.2% mineral oil adjuvant, using 0.7 mL of this solution per plant. Only water + mineral oil adjuvant was applied in the same quantity mentioned above for the non-primed plants. The Se concentration in the priming solution was determined based on previous research, which demonstrated that similar concentrations mitigate various abiotic stresses [26]. Our aims with this selected concentration were a dual effect, consisting of improving the agronomic biofortification process while also combining it with seedlings that were more resistant to different types of stress. Furthermore, this concentration was used because there are no studies with a specific concentration for applying Se to broccoli seedlings to improve the efficiency of agronomic biofortification in the adult stage.

After producing the seedlings, they were transplanted into pots filled with 5 kg of soil (Latossolo Vermelho). According to the Brazilian soil classification system, the soil at the study site was classified as Latossolo Vermelho [38], corresponding to the 0–0.20 m layer, as shown in

Table 1. Chemical, physicochemical, and soil particle size distribution of the soil used.

Attributes	Values
pH in water	4.8
Soil organic matter (g kg−1)	24.9
Clay (g kg−1)	670
Silt (g kg−1)	130
Sand (g kg−1)	200
Total nitrogen (g kg−1)	2.3
Available potassium (mg kg−1)	24.8
Available phosphorus (mg kg−1)	0.4
Exchangeable calcium2+ (cmolc kg−1)	0.4
Exchangeable magnesium2+ (cmolc kg−1)	0.2
Available zinc (mg kg−1)	0.2
Available iron (mg kg−1)	38.0
Available manganese (mg kg−1)	3.4
Available copper (mg kg−1)	1.2
Available boron (mg kg−1)	0.01
Available sulfur (mg kg−1)	2.9
Total selenium (mg kg−1)	0.38

Soil pH was determined in water at a ratio of 1:2.5 (w/v). Soil organic matter was determined using the Walkley–Black method, and the clay, silt, and sand fractions were assessed by the Bouyoucos method. Total nitrogen concentration was determined using the Kjeldahl method. The available concentrations of nutrients were determined by the Mehlich-1 soil test. Calcium and magnesium exchangeable concentrations were extracted by a 1 mol L⁻¹ KCl solution-soil test. The available concentration of boron was determined by the hot-water extraction method, and the available concentration of sulfur was determined by the monocalcium phosphate diluted in acetic acid method. The total concentration of selenium was also determined. All methodologies are described in Teixeira et al. [41]. The Se was determined according to the 3051A USEPA protocol [42].

. This classification is related to Ferralsols [39] and Oxisols in the Soil Taxonomy [40]. Liming was performed on the collected soil to supply calcium and magnesium and correct the soil pH before transplanting. The method used to determine the amount of lime applied was the soil incubation curve with increasing lime rates. Liming was carried out using the soil incubation method for 21 days, maintaining soil moisture at 70% of the maximum water holding capacity (MWHC) and applying calcium (CaCO₃) and magnesium (MgCO₃) carbonates (reagent grade, Synth) in a 3:1 ratio. The pH achieved after soil correction was 5.6 ± 0.03. After pH correction, the soil was dried and sieved (<4 mm).

At the time of planting, the following nutrient quantities were provided: 25, 38, 43.8, 8.20, 0.81, 1.33, 3.66, 0.15, and 1.55 mg kg⁻¹ for nitrogen (N), phosphorus (P), potassium (K), sulfur (S), boron (B), copper (Cu), manganese (Mn), molybdenum (Mo), and iron (Fe), respectively. A granular commercial fertilizer containing 4% N, 6.1% P, and 6.6% K was used for N, P, and K. The remaining K and other nutrients were supplied using K₂SO₄, H₃BO₃, CuSO₄·5H₂O, MnSO₄·7H₂O, Na₂MoO₄, and FeCl₃·6H₂O (reagent grade, Synth). Zinc (Zn) was provided at two concentrations, 5 and 25 mg kg⁻¹, according to the tested treatments, using ZnSO₄·7H₂O (reagent grade, Synth) as the source. Top-dressing with K was performed at 33 and 51 days using 65 mg kg⁻¹ of K per application, with KCl (reagent grade, Synth) as the source. Top-dressing with N was carried out using 65 mg kg⁻¹ of N per application, applying urea (CH₄N₂O) (reagent grade, Synth) 20, 40, and 60 days after transplanting. The nutrient quantities for initial and top-dressing fertilizations were adapted from the recommendations for pot experiments in controlled environments [43].

The transplanted plants were exposed to the treatments described in

Table 2. Description of the agronomic biofortification strategies for the greenhouse experiment.

Acronym	Description
No Se	Control—standard field fertilization, adapted for pots [43]
Planting (50 g Se ha−1) #	Soil application of Se using Se-enriched planting fertilizer to achieve a dose of 50 g Se ha−1
Planting (100 g Se ha−1) #	Soil application of Se using Se-enriched planting fertilizer to achieve a dose of 100 g Se ha−1
Top-dressing (50 g Se ha−1) #	Soil application of Se using Se-enriched top-dressing fertilizer to achieve a dose of 50 g Se ha−1
Top-dressing (100 g Se ha−1) #	Soil application of Se using Se-enriched top-dressing fertilizer to achieve a dose of 100 g Se ha−1
Foliar SeMNF (25 g Se ha−1) †*	Foliar application of Se using foliar Se multi-nutrient fertilizer to achieve a dose of 25 g Se ha−1
Foliar SeMNF (50 g Se ha−1) †*	Foliar application of Se using foliar Se multi-nutrient fertilizer to achieve a dose of 50 g Se ha−1
Foliar SeOrg (25 g Se ha−1) *	Foliar application of Se using foliar Se + organic compounds fertilizer to achieve a dose of 25 g Se ha−1
Foliar SeOrg (50 g Se ha−1) *	Foliar application of Se using foliar Se + organic compounds fertilizer to achieve a dose of 50 g Se ha−1
Foliar Na2SeO4 (25 g Se ha−1) *	Foliar application of Se using foliar sodium selenate to achieve a dose of 25 g Se ha−1
Foliar Na2SeO4 (50g Se ha−1) *	Foliar application of Se using foliar sodium selenate to achieve a dose of 50 g Se ha−1

#: Under field conditions, the proposed supplemental Zn dose for soil application is equivalent to 10 kg Zn ha⁻¹ (~5 mg Zn kg⁻¹ of soil). For pot experiments, this dose should be approximately five times higher, i.e., 25 mg Zn kg⁻¹. The recommended dose for pot experiments is 5 mg Zn kg⁻¹ [43]; ^†: The SeMNF used contained 10.0, 26.2, 149.4, 30, 2.5, 2.5, and 5 g kg⁻¹ of N, P, K, Mg, Se, Zn, and B, respectively. *: The Zn doses applied foliar will follow the Se ratio of the products being tested, meaning that as the Se dose increases, the Zn dose will be adjusted to maintain the Se ratio of the product used.

until the inflorescences were harvested for analysis. The treatments were arranged in a completely randomized design. Since some commercial products already contain Zn (Foliar SeMNF), the amount of this element was standardized. The treatments consisted of applying Se in two doses and three sources, based on previous studies [15,44,45]. Foliar application of Se was carried out in two stages, at 45 days (vegetative phase) and 75 days (reproductive phase) after transplanting. In addition, Se was applied to the soil in two doses and at two different times, using either planting fertilizer (NPK) or top-dressing (N-urea) as the carrier. Based on previous literature recommendations, the additional Zn dose was determined for soil application [44,45]. A control treatment (without Se or supplemental Zn) was also included, and all treatments were applied to seedlings produced with or without priming. Five replications were used, totaling 110 experimental units. The Se doses applied to the soil (50 and 100 g ha⁻¹) were adjusted for soil conditions, equivalent to 0.025 mg kg⁻¹ and 0.05 mg kg⁻¹. For the foliar applications, an average plant population of 20,000 plants per hectare was considered, with a spray volume of 400 L ha⁻¹, such that 20 mL of the spray solution was applied per plant, equivalent to 1.25 mg and 2.5 mg per plant (pot) for the doses of 25 and 50 g ha⁻¹, respectively. Spraying was carried out manually; however, to ensure the applied volume, a manual sprayer with a capacity of 50 mL was used. The volume to be sprayed on each plant was placed (20 mL), and spraying was only completed after the entire volume had been used up. The amount applied (20 mL) was determined to ensure complete coverage of the whole plant without runoff. For this purpose, a previous test was carried out with different application volumes on another set of broccoli plants of the same age, until this optimal concentration was determined.

For the application of Se via planting or top-dressing fertilizer, Se in the form of sodium selenate (Na₂SeO₄) (reagent grade, Sigma Aldrich, St. Louis, MI, USA) was incorporated using the following procedure: Na₂SeO₄, triethanolamine, and an organic dye were first added and mixed in a beaker. After that, planting or top-dressing fertilizer was added to this mixture, and the materials were thoroughly remixed until the urea was uniformly coated and no residual material remained in the beaker. The samples were then dried at room temperature for 24 h and stored for later use. The triethanolamine concentration used was 0.1% (volume/mass). The sodium selenate concentrations used were 0.0082%, 0.0164%, 0.0171%, and 0.0342%, respectively, for the planting fertilizer at the doses of 50 and 100 g Se ha⁻¹, and for the topdressing fertilizer at the doses of 50 and 100 g Se ha⁻¹. This technological package, which involves the production of priming seedlings and their combination with agronomic Se biofortification, is part of a developed patent under number BR1020250158965 [46], such that its reproduction requires authorization.

2.2. Broccoli Harvest

Broccoli harvest was carried out according to maturity, following market standards. The harvest was timed when the lateral florets began to outgrow the central florets of the broccoli head. The harvested samples were washed in distilled water. At the end of the experiment, the following agronomic characteristics were evaluated: production, days to flowering, days to reach the cutting point, head size and weight, floret-to-stem ratio, firmness, moisture, and Se concentrations in raw, blanched, and blanched + cooked florets. Post-harvest operations, including blanching and further preparation, were performed on the collected floret samples [18,19,20].

The raw samples (1/3) that were not blanched were stored in a freezer (−20 °C). For the blanching process (2/3) of the fresh raw floret samples, they were placed in hot water (100 °C) for 60 s at a sample-to-water ratio of 1:10 (w/w). After this process, the samples were drained and placed in ice water (<5 °C) to stop the cooking process and cool the samples. The samples were, again, drained and stored in a freezer [20]. The duration of frozen storage was 30 days. After blanching and freezing, the broccoli was cooked to simulate the final consumer preparation. For cooking, 25 g of blanched and frozen samples were heated in a microwave oven (MT030, Manaus, AM, Brazil) at 800 W for 180 s [19]. After cooking, the samples were immediately frozen and placed in a freezer. Before freezing all samples, floret firmness was measured in Newtons (N) using a Marconi benchtop digital penetrometer (model MA 933, Marconi, Piracicaba, SP, Brazil) equipped with a 1 mm diameter cone tip at a constant speed of 0.01 m min⁻¹.

2.3. Selenium and Zinc in Broccoli Floret

Product analyses were conducted in accordance with international QA/QC protocols and using certified reference materials (CRMs). Both samples and CRMs were analyzed using standard procedures (i.e., digestion with the USEPA 3051A method, followed by analysis with inductively coupled plasma mass spectrometry (ICP-MS) and inductively coupled plasma optical emission spectrometry (ICP-OES)). For determining Se concentrations, samples were dried in a forced-air oven at 45 °C and ground (<0.38 mm) using a stainless-steel mill. Sample digestion was performed in a CEM^® Mars-5 microwave system (CEM Corp., Matthews, NC, USA) according to USEPA Method 3051A [42]. Selenium concentrations in the digested solutions were determined using ICP-MS, while Zn concentrations were quantified using ICP-OES. During the analyses, certified plant material samples (Tomato leaves—NIST SRM 1573a and White clover—BCR 402) were used for QA/QC protocols, with Se recovery rates at 109 ± 9.0% and Zn at 96 ± 4.0%.

Selenium concentration in the fresh mass was calculated based on the Se concentration in the dry mass of the florets, and their moisture content was determined [47] (

Table A1. Effect of different agronomic biofortification strategies with selenium (Se) and processing on moisture of broccoli florets.

Treatment	Se Rate (g ha−1)	Priming	Raw (%)	Blanching (%)	Blanching + Cooking (%)
No Se	0	No	90.4 ± 0.9	91.7 ± 0.8	87.6 ± 0.5
		Yes	88.5 ± 0.6	93.0 ± 0.3	87.2 ± 0.6
Planting	50	No	87.6 ± 0.6	91.3 ± 0.7	88.3 ± 0.9
		Yes	89.6 ± 1.0	93.2 ± 0.4	91.5 ± 0.2
	100	No	90.7 ± 0.6	93.5 ± 0.5	89.5 ± 0.4
		Yes	92.8 ± 0.7	93.1 ± 0.4	90.0 ± 1.1
Top-dressing	50	No	90.8 ± 1.3	91.0 ± 1.0	90.2 ± 0.9
		Yes	88.0 ± 1.0	90.6 ± 0.2	86.9 ± 1.0
	100	No	89.8 ± 1.1	91.6 ± 0.6	88.6 ± 0.6
		Yes	91.9 ± 0.6	92.8 ± 0.1	90.5 ± 0.7
Foliar SeMNF	25	No	89.2 ± 1.0	93.3 ± 0.2	88.9 ± 0.6
		Yes	88.0 ± 0.6	91.7 ± 0.6	88.2 ± 0.4
	50	No	88.5 ± 0.2	93.5 ± 0.5	91.9 ± 0.6
		Yes	86.3 ± 1.3	91.6 ± 0.9	80.3 ± 0.8
Foliar SeOrg	25	No	87.7 ± 0.5	90.1 ± 0.4	88.2 ± 0.3
		Yes	88.0 ± 0.4	92.9 ± 0.8	88.9 ± 0.6
	50	No	88.3 ± 0.4	90.9 ± 0.3	88.4 ± 0.3
		Yes	90.9 ± 0.6	93.2 ± 0.3	90.1 ± 0.8
Foliar Na2SeO4	25	No	89.1 ± 0.6	92.3 ± 0.6	88.2 ± 0.6
		Yes	86.8 ± 0.4	90.6 ± 0.3	87.2 ± 0.7
	50	No	88.7 ± 0.7	93.4 ± 0.2	86.2 ± 0.6
		Yes	86.4 ± 0.7	90.8 ± 0.3	85.4 ± 0.3

Means are shown with standard error (n = 5).

). Using these results and considering the average daily consumption of broccoli (40 g per person per day), the range of Se in fresh mass required to meet 100% of the ideal average daily intake for the human population (60–70 µg per day) and 50% of the adequate intake (30–35 µg per day) [1,2,3,4,48] was calculated. Selenium concentration in fresh mass values was also used to assess Se losses during the blanching and cooking processes.

2.4. Statistical Analysis

All statistical analyses were conducted using R software version 4.5.0 [49], with the following packages: stats 4.5.0, Metrics version 0.1.4, agricolae version 1.3.7, factoextra version 1.0.7, and FactoMineR version 2.12 [49,50,51,52,53]. Treatment means were grouped using the Scott–Knott test (p < 0.05) after confirming that the basic assumptions of analysis of variance (normality, homoscedasticity, additivity, and independence of residuals) were met and achieving significance in the F-test (p < 0.05). A preliminary analysis was conducted using the “Anova function”, followed by comparisons between each processing method and the raw samples for each biofortification strategy using multiple comparisons with the “ghlt function” [54] to differentiate the effects of various post-harvest processing methods on Se concentration.

3. Results

3.1. Agronomic Characteristics

All studied agronomic characteristics of broccoli, except head diameter (HD), were affected by the interaction between the studied factors (p < 0.05,

Table 3. Effect of different agronomic biofortification strategies with selenium (Se) on agronomic characteristics of broccoli.

Treatment	Se Rate (g ha−1)	Priming	DF (Days)	DH (Days)	TP (g plant−1)	HD (cm plant−1)	FW (g plant−1)	SW (g plant−1)	F:S Ratio (g g−1)
No Se	0	No	69 ± 1 aA	95 ± 1 aA	166 ± 16 aA	169 ± 6 aA	130 ± 14 aA	36 ± 3 aA	3.7 ± 0.3 aA
		Yes	65 ± 3 bB	90 ± 3 bC	165 ± 8 aB	175 ± 7 aA	127 ± 8 aB	39 ± 2 aA	3.3 ± 0.3 bA
Planting	50	No	61 ± 2 bB	87 ± 2 aB	177 ± 17 aA	163 ± 8 aA	137 ± 15 aA	40 ± 3 aA	3.4 ± 0.3 bA
		Yes	65 ± 4 aB	87 ± 4 aC	201 ± 24 aA	178 ± 9 aA	158 ± 19 aA	43 ± 6 aA	3.7 ± 0.3 aA
	100	No	66 ± 1 aB	92 ± 1 aA	167 ± 17 aA	159 ± 8 aB	120 ± 14 aA	47 ± 3 aA	2.5 ± 0.2 bB
		Yes	68 ± 2 aA	95 ± 3 aB	131 ± 9 aB	140 ± 4 aB	98 ± 9 aB	32 ± 2 bB	3.1 ± 0.3 aB
Top-dressing	50	No	64 ± 1 aB	85 ± 2 bB	177 ± 17 aA	168 ± 13 aA	132 ± 13 aA	44 ± 5 aA	3.0 ± 0.1 bB
		Yes	65 ± 2 aB	90 ± 3 aC	186 ± 21 aA	168 ± 6 aA	140 ± 16 aA	46 ± 5 aA	3.1 ± 0.2 aB
	100	No	66 ± 3 aB	91 ± 3 aA	185 ± 13 aA	172 ± 4 aA	142 ± 10 aA	43 ± 3 aA	3.3 ± 0.1 bA
		Yes	67 ± 4 aA	91 ± 4 aC	187 ± 12 aA	178 ± 7 aA	146 ± 10 aA	42 ± 3 aA	3.5 ± 0.3 aA
Foliar SeMNF	25	No	69 ± 4 aA	93 ± 4 aA	166 ± 15 aA	170 ± 14 aA	127 ± 14 aA	39 ± 2 aA	3.2 ± 0.4 bA
		Yes	62 ± 2 bB	88 ± 3 aC	186 ± 9 aA	169 ± 7 aA	147 ± 7 aA	39 ± 2 aA	3.8 ± 0.2 aA
	50	No	69 ± 1 aA	94 ± 2 aA	184 ± 15 aA	168 ± 9 aA	138 ± 14 aA	46 ± 3 aA	3.1 ± 0.4 bB
		Yes	66 ± 3 aB	89 ± 3 bC	204 ± 20 aA	171 ± 8 aA	157 ± 16 aA	47 ± 5 aA	3.4 ± 0.2 aB
Foliar SeOrg	25	No	71 ± 2 aA	96 ± 2 aA	140 ± 15 aA	134 ± 15 aB	103 ± 12 aA	37 ± 3 aA	2.8 ± 0.1 bB
		Yes	68 ± 4 aA	94 ± 5 aB	157 ± 20 aB	147 ± 13 aB	118 ± 16 aB	39 ± 4 aA	3.0 ± 0.1 aB
	50	No	66 ± 0 aB	92 ± 1 aA	193 ± 4 aA	171 ± 5 aA	149 ± 2 aA	44 ± 3 aA	3.4 ± 0.2 bA
		Yes	65 ± 2 aB	88 ± 2 aC	152 ± 14 bB	162 ± 10 aA	119 ± 12 aB	32 ± 3 bB	3.7 ± 0.2 aA
Foliar Na2SeO4	25	No	65 ± 2 aB	89 ± 2 aB	193 ± 10 aA	169 ± 5 aA	147 ± 10 aA	46 ± 2 aA	3.2 ± 0.2 bB
		Yes	65 ± 4 aB	90 ± 4 aC	177 ± 10 aA	159 ± 9 aA	134 ± 7 aA	43 ± 4 aA	3.2 ± 0.2 aB
	50	No	64 ± 1 bB	87 ± 2 bB	186 ± 12 aA	167 ± 6 aA	141 ± 11 aA	46 ± 2 aA	3.1 ± 0.2 bB
		Yes	71 ± 1 aA	101 ± 2 aA	127 ± 12 bB	156 ± 7 aA	97 ± 10 bB	30 ± 2 bB	3.2 ± 0.3 aB

DF: days to flowering; DH: days to harvest; TP: total fresh head production; HD: head diameter; FW: fresh weight of the floret; SW: stalk weight; F:S ratio: the floret-to-stalk ratio of the broccoli samples. Means are shown with standard error (n = 5). Uppercase letters indicate significant differences among different Se treatments within each priming condition, while lowercase letters indicate significant differences between priming and non-priming treatments within each Se treatment (Scott–Knott test, p < 0.05).

). Seedlings with priming reduced days to flowering (DF) by approximately 6 days for the following biofortification strategies: no Se, Planting (50 g Se ha⁻¹), and Foliar SeMNF (25 g Se ha⁻¹) (Table 3). However, for Foliar Na₂SeO₄ (50 g Se ha⁻¹), the priming treatment increased DF by 6.6 days. For the other strategies, there was no difference in DF between the priming treatment and seedlings without priming. In cultivation conditions where plants were produced without the priming effect, the DF were similar among treatments No Se, Foliar SeMNF (25 g Se ha⁻¹), Foliar SeMNF (50 g Se ha⁻¹), and Foliar SeOrg (25 g Se ha⁻¹) (p > 0.05). The other treatments hastened the flowering time by an average of 4.9 days (p < 0.05). However, in cultivation conditions using priming seedlings, the treatments Planting (100 g Se ha ha⁻¹), Top-dressing (100 g Se ha ha⁻¹), Foliar SeOrg (25 g Se ha ha⁻¹), and Foliar Na₂SeO₄ (50 g Se ha⁻¹) showed a longer DF compared with the other treatments, with an average increase of 4.0 days.

Seedlings with priming achieved an average reduction of approximately 5.5 days to harvest (DH) for the biofortification strategies, which included both Se application and foliar application of Se with a multi-nutrient fertilizer (50 g Se ha⁻¹). However, for top-dressing (50 g Se ha⁻¹) and Foliar Na₂SeO₄ (50 g Se ha⁻¹), the priming treatment increased DH by 5 and 13.9 days, respectively. For the other biofortification strategies, there was no effect of using seedlings with priming compared with seedlings without priming. In the cultivation of seedlings with priming, the treatments No Se, Planting (100 g Se ha⁻¹), Top-dressing (100 g Se ha⁻¹), Foliar SeMNF (25 g Se ha⁻¹), Foliar SeMNF (50 g Se ha⁻¹1), Foliar SeOrg (25 g Se ha⁻¹), and Foliar SeOrg (50 g Se ha⁻¹) showed a longer DH compared with the other treatments, with an average increase of 6.6 days. In cultivation conditions using seedlings with priming, the treatment with Foliar Na₂SeO₄ (50 g Se ha⁻¹) had the longest DH (100.5 days), followed by the Planting (100 g Se ha⁻¹) and Foliar SeOrg (25 g Se ha⁻¹) treatments (average of 94.8 days), which had longer DH compared with the other treatments (average of 89 days).

Seedlings produced with priming resulted in a reduction in total fresh head production (TP) by 22% for the biofortification strategy Foliar SeOrg (50 g Se ha⁻¹) and by 32% for Foliar Na₂SeO₄ (50 g Se ha⁻¹), as shown in Table 3. For the other strategies, there was no difference in TP between the seedlings with priming and the seedlings without priming. For seedlings produced without priming, the tested biofortification strategies had no effect on TP. However, for the cultivation of seedlings with priming, the treatments Planting (50 g Se ha⁻¹), Top-dressing (50 g Se ha⁻¹), Top-dressing (100 g Se ha⁻¹), Foliar SeMNF (25 g Se ha⁻¹), Foliar SeMNF (50 g Se ha⁻¹), and Foliar Na₂SeO₄ (25 g Se ha⁻¹) increased TP by approximately 30% compared with No Se and the other treatments.

The head diameter of broccoli was affected solely by biofortification strategies (Table 3). The biofortification strategies, Planting (100 g Se ha⁻¹) and Foliar SeOrg (25 g Se ha⁻¹), reduced the HD by an average of 14% compared with the other treatments.

Seedlings produced with priming resulted in a 31% reduction in FW for the biofortification strategy using Foliar Na₂SeO₄ (50 g Se ha⁻¹), as presented in Table 3. For the other strategies, there was no difference in FW between the seedlings with priming and the seedlings without priming. The biofortification strategies did not affect plant production for seedlings produced without priming. However, for the cultivation of seedlings with priming, the treatments Planting (50 g Se ha⁻¹), Top-dressing (50 g Se ha⁻¹), Top-dressing (100 g Se ha⁻¹), Foliar SeMNF (25 g Se ha⁻¹), Foliar SeMNF (50 g Se ha⁻¹), and Foliar Na₂SeO₄ (25 g Se ha⁻¹) increased FW by approximately 31% compared with No Se and the other treatments.

Seedlings produced with priming resulted in a 30% reduction in SW for the biofortification strategies of Planting (100 g Se ha⁻¹), Foliar SeOrg (50 g Se ha⁻¹), and Foliar Na₂SeO₄ (50 g Se ha⁻¹), as shown in Table 3. For the other strategies, there was no difference in production between seedlings with priming and those without priming. The biofortification strategies did not affect the SW for seedlings produced without priming. However, for the cultivation of seedlings with priming, the treatments Planting (100 g Se ha⁻¹), Foliar SeOrg (50 g Se ha⁻¹), and Foliar Na₂SeO₄ (50 g Se ha⁻¹) reduced the SW by approximately 25% compared with the other treatments.

For the F:S ratio, there was no interaction between the studied factors (p > 0.05), with only the effect of the tested biofortification strategies observed (Table 3). Seedling priming increased the F:S ratio of broccoli under biofortification strategies, although it decreased the F:S ratio in No Se broccoli (p < 0.05, Table 3).

No Se and the biofortification strategies Planting (50 g Se ha⁻¹), Top-dressing (100 g Se ha⁻¹), Foliar SeMNF (25 g Se ha⁻¹), and Foliar SeOrg (50 g Se ha⁻¹) increased the F:S ratio by an average of 15% compared with the other treatments, despite priming.

The firmness of raw, blanched, and cooked florets (73.3 N ± 1.4, 58.7 N ± 1.2, and 16.3 N ± 0.5, respectively) was not affected by the adopted biofortification strategies (p > 0.05) (

Table 4. Effect of different agronomic biofortification strategies with selenium (Se) and processing on firmness (N) of broccoli florets.

Treatment	Se Rate (g ha−1)	Priming	Raw (N)	Blanching (N)	Blanching + Cooking (N)
No Se	0	No	75.5 ± 4.6	55.1 ± 4.1	19.1 ± 2.0
		Yes	62.2 ± 2.7	64.0 ± 4.6	15.2 ± 2.0
Planting	50	No	76.4 ± 3.3	67.7 ± 3.9	15.6 ± 1.9
		Yes	72.3 ± 5.3	59.7 ± 5.2	14.3 ± 1.6
	100	No	71.2 ± 5.3	54.6 ± 4.6	16.6 ± 1.9
		Yes	62.9 ± 5.5	57.5 ± 3.4	16.4 ± 1.4
Top-dressing	50	No	79.0 ± 7.7	51.0 ± 4.3	19.0 ± 1.1
		Yes	83.1 ± 5.3	69.6 ± 4.6	16.7 ± 1.5
	100	No	71.7 ± 3.0	59.7 ± 3.4	22.0 ± 0.8
		Yes	69.4 ± 8.0	55.1 ± 2.5	16.7 ± 0.9
Foliar SeMNF	25	No	71.7 ± 8.8	56.0 ± 2.6	14.6 ± 0.9
		Yes	72.5 ± 6.7	54.1 ± 6.0	20.2 ± 1.3
	50	No	71.8 ± 5.4	59.2 ± 4.2	18.2 ± 1.5
		Yes	88.4 ± 5.9	58.1 ± 7.6	12.4 ± 0.9
Foliar SeOrg	25	No	76.5 ± 2.3	61.9 ± 3.7	14.2 ± 0.8
		Yes	60.1 ± 5.7	53.3 ± 7.4	16.7 ± 2.4
	50	No	74.6 ± 3.8	56.9 ± 7.5	16.6 ± 1.4
		Yes	73.9 ± 4.2	52.2 ± 3.1	15.0 ± 1.7
Foliar Na2SeO4	25	No	78.7 ± 3.8	59.2 ± 3.7	14.9 ± 1.3
		Yes	71.5 ± 6.2	57.1 ± 3.2	16.8 ± 1.0
	50	No	77.6 ± 7.8	72.1 ± 2.2	13.5 ± 0.8
		Yes	72.4 ± 5.6	57.5 ± 4.5	13.7 ± 1.1
Treatments mean			73.3 ± 1.4 A	58.7 ± 1.2 B	16.3 ± 0.50 C

Means are shown with standard error (n = 5). Different italic uppercase letters in the last line compare the firmness means of the florets for all treatments under each post-harvest condition evaluated (Scott–Knott test, p < 0.05).

). The blanching and subsequent cooking reduced the firmness of broccoli florets by 19.9% and 77.8%, respectively, compared with raw florets (Table 4).

3.2. Selenium Concentration in Florets

As the study focused primarily on Se biofortification, with little effect observed on Zn, data related to Zn concentration in florets based on dry mass are presented in

Table A2. Effect of different agronomic biofortification strategies with selenium (Se) and processing on zinc concentration of broccoli dry florets.

Treatment	Se Rate (g ha−1)	Priming	Raw (mg kg−1)	Blanching (mg kg−1)	Blanching + Cooking (mg kg−1)
No Se	0	No	48.1 ± 2.3	50.0 ± 7.0	38.6 ± 3.1
		Yes	46.4 ± 3.3	73.5 ± 4.9	42.7 ± 1.9
Planting	50	No	49.8 ± 1.9	76.3 ± 9.5	51.0 ± 4.9
		Yes	60.2 ± 1.7	91.0 ± 1.8	65.1 ± 1.1
	100	No	60.9 ± 3.4	83.6 ± 7.9	60.0 ± 4.5
		Yes	64.1 ± 3.0	68.6 ± 5.2	42.5 ± 3.6
Top-dressing	50	No	49.9 ± 6.0	52.4 ± 8.5	38.2 ± 2.8
		Yes	48.3 ± 3.1	61.6 ± 5.6	39.9 ± 2.1
	100	No	49.1 ± 5.0	60.7 ± 5.0	36.2 ± 1.3
		Yes	65.4 ± 3.3	65.4 ± 4.2	47.4 ± 5.1
Foliar SeMNF	25	No	46.9 ± 5.4	70.6 ± 4.2	43.9 ± 1.8
		Yes	32.2 ± 2.7	48.8 ± 4.0	34.5 ± 2.3
	50	No	52.1 ± 2.7	77.1 ± 9.6	62.8 ± 7.1
		Yes	49.9 ± 2.2	76.7 ± 10.1	33.8 ± 2.9
Foliar SeOrg	25	No	51.0 ± 5.0	50.9 ± 4.3	48.2 ± 2.9
		Yes	51.2 ± 3.7	79.5 ± 7.7	45.9 ± 3.3
	50	No	35.4 ± 0.6	31.8 ± 2.7	29.4 ± 0.9
		Yes	41.6 ± 3.1	56.7 ± 1.4	34.5 ± 1.9
Foliar Na2SeO4	25	No	42.2 ± 2.5	52.9 ± 2.4	33.1 ± 1.5
		Yes	49.5 ± 3.0	53.8 ± 4.7	39.4 ± 2.3
	50	No	45.8 ± 2.3	60.5 ± 3.8	31.8 ± 2.7
		Yes	39.9 ± 2.8	54.5 ± 2.4	28.3 ± 1.1

Means are shown with standard error (n = 5).

. For Se concentration in raw florets based on dry mass, there was an interaction between the studied factors (p < 0.05). Seedlings produced with priming promoted higher Se concentration in the raw florets (dry mass) under biofortification strategies using Top-dressing (100 g Se ha⁻¹) (+184%), Foliar SeMNF (50 g Se ha⁻¹) (+67%), Foliar SeOrg (50 g Se ha⁻¹) (+45%), Foliar Na₂SeO₄ (25 g Se ha⁻¹) (+42%), and Foliar Na₂SeO₄ (50 g Se ha⁻¹) (+166%), reaching values of 3.5, 23.1, 13.8, 6.8, and 18.4 mg kg⁻¹ of Se in dry mass, respectively (Figure 1). For the other strategies, there was no difference in Se concentration in the dry mass when comparing seedlings produced with and without priming.

For seedlings produced without priming, the Foliar SeMNF (50 g Se ha⁻¹) biofortification strategy resulted in the highest Se concentration in the dry mass of raw florets (13.9 mg kg⁻¹), followed by Foliar SeMNF (25 g Se ha⁻¹) and Foliar SeOrg (50 g Se ha⁻¹) (10.1 and 9.5 mg kg⁻¹, respectively), and higher than other treatments. In contrast, for seedlings produced with priming, the highest Se concentration values in dry mass followed the descending order: Foliar SeMNF (50 g Se ha⁻¹) (23.1 mg kg⁻¹) > Foliar Na₂SeO₄ (50 g Se ha⁻¹) (18.4 mg kg⁻¹) > Foliar SeOrg (50 g Se ha⁻¹) (13.8 mg kg⁻¹) > Foliar SeMNF (25 g Se ha⁻¹) (8.8 mg kg⁻¹) > Foliar Na₂SeO₄ (25 g Se ha⁻¹) = Foliar SeOrg (25 g Se ha⁻¹) (average of 6.0 mg kg⁻¹) > Top-dressing (100 g Se ha⁻¹) (3.5 mg kg⁻¹ > No Se = Planting (50 g Se ha⁻¹) = Planting (100 g Se ha⁻¹) = Top-dressing (50 g Se ha⁻¹) (average of 0.33 mg kg⁻¹).

For Se concentration based on dry mass in florets after blanching, there was also an interaction between the factors studied (p < 0.05). Seedlings with priming enhanced the increase in Se concentration in the dry mass of blanched florets, with a rise in biofortification strategies using Foliar SeMNF (25 g Se ha⁻¹) (+25%), Foliar SeMNF (50 g Se ha⁻¹) (+143%), Foliar SeOrg (50 g Se ha⁻¹) (+58%), and Foliar Na₂SeO₄ (50 g Se ha⁻¹) (+56%) compared with the same strategies without the use of seedlings with priming, reaching values of 10.1, 32.3, 16.2, and 22.0 mg kg⁻¹ of Se in dry mass of blanched floret, respectively (Figure 1). For the other strategies, the Se concentration in the dry mass of blanched florets did not differ between seedlings produced with and without priming. For seedlings produced without priming, the Foliar SeMNF (50 g Se ha⁻¹) (14.1 mg kg⁻¹) and Foliar Na₂SeO₄ (50 g Se ha⁻¹) (13.3 mg kg⁻¹) biofortification strategies resulted in the highest Se concentration in the dry mass of blanched florets, followed by Foliar SeMNF (25 g Se ha⁻¹), Foliar SeOrg (50 g Se ha⁻¹), and Foliar Na₂SeO₄ (25 g Se ha⁻¹) (8.8, 8.1, and 10.2 mg kg⁻¹, respectively), values higher than those of the other treatments. For seedlings produced with priming, the highest Se concentration values in the dry mass of blanched florets followed the descending order: Foliar SeMNF (50 g Se ha⁻¹) (32.3 mg kg⁻¹) > Foliar Na₂SeO₄ (50 g Se ha⁻¹) (22.0 mg kg⁻¹) > Foliar SeOrg (50 g Se ha⁻¹) (16.2 mg kg⁻¹) > Foliar SeMNF (25 g Se ha⁻¹) (10.1 mg kg⁻¹) = Foliar Na₂SeO₄ (25 g Se ha⁻¹) (9.3 mg kg⁻¹), with these treatments being highest than the others treatments.

For Se concentration based on dry mass in blanched and cooked florets, there was also an interaction between the factors studied (p < 0.05). Seedlings produced with priming enhanced the increase in Se concentration in the dry mass of cooked florets, with a rise in biofortification strategies using Foliar SeMNF (50 g Se ha⁻¹) (+22%), Foliar SeOrg (50 g Se ha⁻¹) (+43%), Foliar Na₂SeO₄ (25 g Se ha⁻¹) (+45%), and Foliar Na₂SeO₄ (50 g Se ha⁻¹) (+169%), reaching values of 19.2, 14.1, 5.85, and 14.13 mg kg⁻¹ of Se in dry mass, respectively (Figure 1). For the other strategies, Se concentration in cooked florets did not increase when comparing seedlings produced with and without priming. For seedlings produced without priming, the Foliar SeMNF (50 g Se ha⁻¹) (15.69 mg kg⁻¹) biofortification strategy resulted in the highest Se concentration in the dry mass of cooked florets, followed by Foliar SeMNF (25 g Se ha⁻¹) and Foliar SeOrg (50 g Se ha⁻¹) (9.2 and 9.8 mg kg⁻¹, respectively), values higher than those of the other treatments. For seedlings produced with priming, the highest Se concentration values in the dry mass of cooked florets followed the descending order: Foliar SeMNF (50 g Se ha⁻¹) (19.2 mg kg⁻¹) > Foliar Na₂SeO₄ (50 g Se ha⁻¹) (14.1 mg kg⁻¹) = Foliar SeOrg (50 g Se ha⁻¹) (14.1 mg kg⁻¹), with these treatments being superior to the others.

3.3. Effect of Processing on Se in Florets and Its Recommendation in the Diet

In general, based on the adequate consumption of 40 g of broccoli in the human diet and a daily Se requirement for humans of 60–70 µg Se day⁻¹ [1,2,3,4,48], the Se concentration in fresh broccoli mass needed to meet this demand would be 1.5 to 1.75 mg kg⁻¹ of Se in fresh mass. Thus, it is observed that the biofortification strategies Foliar SeMNF (50 g Se ha⁻¹) (with and without priming), Foliar SeOrg (50 g Se ha⁻¹), and Foliar Na₂SeO₄ (50 g Se ha⁻¹) (with priming) can provide Se amounts exceeding the required daily intake for humans (Figure 2). When evaluating the supply of 50% of the daily Se requirement for humans (30 to 35 µg Se day⁻¹), in addition to the strategies mentioned above, the biofortification strategies Foliar SeMNF (25 g Se ha⁻¹) (with and without priming), Foliar SeOrg (50 g Se ha⁻¹) (without priming), Foliar Na₂SeO₄ (25 g Se ha⁻¹) (with priming), and Foliar Na₂SeO₄ (50 g Se ha⁻¹) (without priming) are capable of meeting 50% of this requirement (Figure 2). Thus, it is observed that the application of SeMNF and the production of seedlings with priming enhance the Se absorption process and consequently produce florets with higher nutritional values.

Comparing the different processing methods with raw florets, Se losses during processing (blanching and blanching + cooking) occurred only in florets with the highest Se concentrations in fresh mass, specifically with the Foliar Na₂SeO₄ (50 g Se ha⁻¹) (with priming) strategy (Figure 2). Processing did not result in Se loss in the other agronomic biofortification strategies studied.

4. Discussion

Based on our data, it was possible to define effective Se strategies for biofortifying broccoli florets, with minimal impact from the preparation process (Figure 1 and Figure 2). Selenium biofortification emerges as an effective strategy to improve the nutritional quality of food and address the deficiency of this essential element for human health [3,5,7]. However, biofortification strategies with Se are typically only adopted by producers when they do not result in production losses [6,7]. Among the strategies tested, it was found that the treatments leading to the highest Se concentration in the broccoli florets, using seedlings with priming and applying sodium selenate or Se + organic compounds at the same dose (50 g Se ha⁻¹), reduced total broccoli production and floret production by an average of 27% and 30%, respectively, likely due to possible Se toxicity (Table 3 and Figure 1).

Selenium in the plant can promote the substitution of sulfur in the formation of amino acids (cysteine and methionine), forming selenomethionine and selenocysteine [1,48]. However, dysfunctional proteins are formed when this substitution is high, and glutathione synthesis interferes, leading to oxidative stress and reducing crop production [55,56,57]. Due to this effect of Se on plant metabolism, the lifecycle or shelf life of the harvested product may be shortened or extended depending on the Se dose and application strategy [12,58]. In our study, this effect was observed in the broccoli lifecycle, which was either shortened or extended depending on the strategy used. However, this change in the lifecycle was not directly related to broccoli production or the firmness of the harvested and processed product (Table 3 and Table 4).

As long as the plant’s lifecycle changes and there is no loss in productivity, incorporating Se into selenomethionine and selenocysteine is highly desirable, as Se in organic forms is more bioavailable when ingested [4,8]. Additionally, when Se is consumed in organic forms through plants, it is more effective in combating human diseases, as demonstrated in several studies [4,8,10]. For example, studies conducted on rats have shown that, compared with mineral sources of Se, a diet including high-Se broccoli protected animals from colon cancer [10]. One of the treatments that promoted the highest Se concentration in plants without compromising their productivity was the application of higher doses (50 g Se ha⁻¹) via SeMNF, with the effect maximized when plants were subjected to priming. The observed effect of Se application in combination with a multi-nutrient fertilizer is due to other elements that enhance Se absorption and incorporation in plants [12,36]. For instance, studies involving the foliar application of SeMNF have shown improved Se absorption and accumulation in tomato fruits [12]. Moreover, research on soybeans indicated that SeMNF application not only increased Se concentration but also boosted soybean production, which is directly related to nitrogen metabolism [36].

When plants were produced without priming, the effect of SeMNF application in enhancing Se absorption and concentration in broccoli florets was significant, as SeMNF outperformed other foliar-applied products at the highest tested Se dose (50 g Se ha⁻¹) (Figure 1). However, it was observed that when plants were produced with priming, the agronomic biofortification process was maximized compared with seedlings produced without priming (Figure 1). The priming effect can be defined as the application of an agent that triggers a metabolic response in the plant at later stages [32]. Studies show that when plants are subjected to early application of elements, they can develop a metabolic apparatus capable of maximizing the absorption of these elements in later stages (adult plants), thereby increasing the efficiency of the applied elements [34,59]. Selenium priming is an essential strategy for mitigating various stresses, thereby maintaining plant development under adverse conditions [31,33,35]. In our study, the prior application of Se during seedlings significantly increased Se concentrations in broccoli florets at later stages, mainly when higher doses of Se were applied via foliar sprays (Figure 1). Although this effect is not well studied for Se, it has been shown for zinc that using seed priming maximized the agronomic biofortification of maize in adult plants [59]. Since the Se absorption process occurs by transporters like that of S [48,57,60,61], when Se is applied mainly in the form of selenate, it is absorbed by sulfate carriers (SULTRs). The activation process that occurs can be evaluated by the expression of the enzyme APS (adenosine 5’-phosphosulfate). Later, selenate is reduced by the enzyme APR (Adenosine 5’-phosphosulfate reductase) [48,58,62]. Therefore, new studies must be carried out to evaluate how the use of priming seedlings combined with the agronomic biofortification process can change the expression of these genes.

Compared with soil fertilization, our study found that foliar application was more effective in increasing Se concentration in broccoli florets (Figure 1). This result occurs because Se is susceptible to various reactions that render it unavailable when applied to the soil, consequently reducing the efficiency of the agronomic biofortification strategy, which requires higher doses than foliar application, as shown in several studies [5,6,63].

Similarly to our study, other studies have demonstrated the potential of broccoli in agronomic biofortification processes with selenium [13,14,15,29]. This condition is attributed to the high level of sulfur compounds in broccoli, and the metabolism of Se is analogous to that of sulfur [16,17,61,64]. In a study conducted on broccoli under field conditions, it was demonstrated that the application of 50 and 100 g ha⁻¹ of Se using selenite and selenate in different cultivars (Belstar and Legend) increased Se concentration in broccoli heads, with no differences observed between the tested sources, only between the doses [15]. In this study, regardless of the cultivar, the application of 50 g of Se ha⁻¹ reached values of 3.15 mg kg⁻¹ in dry mass, and the dose of 100 g of Se ha⁻¹ reached values of 6.7 mg kg⁻¹ in dry mass. Compared with this study, the concentrations in the florets from our study were higher; however, it is essential to highlight the difference in conditions (field conditions vs. greenhouse conditions).

Additionally, in a study involving broccoli grown in a greenhouse, the application of 15 mL of a solution containing 0.15% Se enabled the achievement of Se levels in boiled florets of 54.0 mg kg⁻¹ [13]. However, the production in that study was only 98.8 g of florets per plant, which is approximately 25% lower than the production in our study, where the average production was 132 g of florets per plant. This explains the higher Se concentration in the previous study, which was due to the dilution effect of elements caused by greater plant growth in our experiment.

In a study conducted with broccoli grown in a greenhouse, considering an average consumption of 100 g of boiled florets, agronomic biofortification of broccoli could provide an amount ranging from 142 to 666 µg of Se per day [13]. However, it is important to highlight that this consumption is much higher than the adequate intake (40 g) and the global average consumption of around 10 g of broccoli per day [65]. In our study, we also demonstrated that broccoli can be biofortified to meet the Se requirements of the human population. Compared with the mentioned studies, we observed that Se concentration ranges from 0.21 to 32.3 mg kg⁻¹ in dry mass, varying according to the adopted biofortification strategy. Based on an adequate broccoli consumption of 40 g, Se concentrations in fresh broccoli required to meet the daily recommended intake of Se (60–70 µg day⁻¹) would be 1.5–1.75 mg kg⁻¹ [1,2,3,4,48]. It was observed that strategies including the application of 50 g Se ha⁻¹ using Foliar SeMNF and seedlings without priming (1.32 mg of Se kg⁻¹—fresh mass) and the strategy using seedlings with priming plus the application of 50 g Se ha⁻¹ using foliar Na₂SeO₄ (2.21 mg of Se kg⁻¹—fresh mass), foliar SeOrg (1.72 mg of Se kg⁻¹—fresh mass), and foliar SeMNF with seedlings without priming (2.07 mg of Se kg⁻¹—fresh mass) were effective to meet the aforementioned daily Se requirement.

The addition of the mentioned strategies successfully met this requirement, with the processing not significantly affecting Se loss during processing, except for the highest Se concentration observed (foliar application of Na₂SeO₄ at 50 g ha⁻¹), where cooking and blanching resulted in a 21% loss of Se in Se-biofortified broccoli. Compared with raw products, broccoli processing affects its quality [18,19,20]. In agronomic biofortification, the processing of the harvested product can lead to losses in Se concentration in the edible part [22]. Therefore, it is crucial to evaluate the concentration of Se in the final product to be consumed, which mainly depends on the location where Se is present (mineral or organic forms) in the harvested part and the preparation method used [66,67]. The ability of plants to incorporate Se absorbed into organic forms of Se is limited, a limitation that occurs mainly when plants accumulate higher levels of this element, but this capacity also depends on the plant species. These forms of Se help explain Se losses in the processing of edible parts [11,48,57,67,68].

Two main mechanisms help explain why mineral forms of Se present in foods are more easily lost in bleaching and cooking processes, when compared to forms of Se that are absorbed and incorporated into organic compounds. The first is that selenate in mineral forms (selenate and selenite) is highly soluble in water and can therefore be lost through leaching during washing and bleaching processes [67,68]. The second mechanism is that due to the heating, these mineral forms are more likely to be converted into volatile compounds such as dimethylselenide (DMSe), promoting losses by volatilization [67,68]. This condition occurs because, while inorganic forms of Se tend to remain stable, forms of selenomethionine and selenocysteine can be lost through volatilization due to increased temperatures during various processing methods of edible parts [68]. However, in potatoes, it has been shown that boiling, compared to other preparation methods such as frying, results in lower Se losses [68]. In contrast to these results, it was observed that such losses were not significant for the blanching and blanching + cooking processes of broccoli (Figure 2). Similarly to our study, which evaluated the boiling process in Se-biofortified cereals and soybeans, it was found that the loss of Se was small, at less than 8.1% [66].

Thus, our study indicates how priming seedling production can maximize the applied doses of Se, enhancing the agronomic biofortification process. In addition, studies related to Se transporters and Se metabolism involving its absorption and incorporation should be conducted to assess how prior Se exposure in seedlings can alter plant metabolism when Se is applied at the adult stage. Nevertheless, further field studies should be performed to validate this technology and to promote research on Se bioaccessibility in the produced florets, aiming to determine which fraction of Se is bioavailable. We emphasize that, despite our promising results, new studies should be conducted to verify the formation of organic forms of Se due to the strategies employed and to investigate how these forms affect the process of Se loss during processing, as well as their bioaccessibility and bioavailability to humans.

5. Conclusions

The foliar application of selenium (Se) is more effective than soil application for producing Se-biofortified florets. Applying higher doses of Se (50 g Se ha⁻¹) combined with a multi-nutrient fertilizer is more effective than Se fertilization using pure Se salts, such as sodium selenate. The application of Se during the seedling stage (priming effect) can increase Se uptake in adult plants, particularly when higher doses of Se are applied via foliar application; consequently, there is an increase in Se concentration in biofortified florets. Therefore, using priming in seedlings enhances the efficiency of Se foliar fertilization in higher doses (50 g Se ha⁻¹) that are later applied to adult plants. The processing of broccoli, including blanching and cooking, does not lead to significant Se losses in biofortified florets, except for a Se concentration of 2.1 mg kg⁻¹ based on the fresh mass of broccoli. Considering the adequate intake of 60–70 µg Se day⁻¹ per person, strategies for producing broccoli florets that involve using seedlings with priming and foliar Se application effectively produce functional broccoli florets to meet this dietary demand, given the consumption of 40 g of broccoli. Despite the promising results, validating these techniques under field conditions in commercial broccoli crops remains essential. Doing so will enable better adjustment of Se doses to maximize biofortification and provide farmers with reliable, reproducible agronomic recommendations. We emphasize that although priming is a promising technique for increasing the agronomic biofortification process, there is a need for studies on genes related to Se absorption and assimilation to understand how this activation occurs at the metabolic level in plants.