Effect of Plant Biostimulants on Beetroot Seed Productivity, Germination, and Microgreen Quality

Abstract

Seed productivity and quality are the bases of modern agriculture. To determine the optimal conditions in terms of seed production and quality, the effect of foliar plant biostimulant treatments (at the beginning and in the middle of the peduncle formation phase and at the beginning of flowering) based on amino acids (Multimolig M and Aminosil), silicon (Si) (Siliplant), selenium (nano-Se), a Rhodotorula glutinis soil yeast formulation, and a fertilizer (Wuxal Macromix), plus an untreated control (only water-sprayed plants), were assessed on Beta vulgaris seed plants grown in an open field in the Moscow region in 2022–2023. Silicon and nano-Se foliar supply led to the highest seed production and viability, as well as positively affecting the yield and quality of the microgreens produced from the latter seeds. Despite the stability of the size distribution of small- and large-sized seeds, only the application of Si increased the occurrence of the large-sized seed class by up to 53%, while R. glutinis fostered a homogenous distribution of seeds among the different diameter classes. The application of all of the biostimulants, except R. glutinis, provided a decrease in oxidative stress in the seeds (reflected in a significant reduction in proline levels), especially for the small-sized seed class, with the highest beneficial effects being caused by Aminosil and Siliplant. All of the treatments were beneficial in terms of chlorophyll and betalain pigment accumulation but did not significantly affect the microgreens’ antioxidant status. The beneficial effect of the biostimulants revealed provides the basis for beetroot seed production and quality improvements to meet the requirements of the Sustainable Development Goals of the United Nations aiming to fight hunger and improve human health and well-being.

1. Introduction

Beetroot (Beta vulgaris L.) is one of the most popular crops among root vegetables, valuable for its high nutritional and medicinal properties both as mature plants [1,2,3] and microgreens [4,5,6]. To date, enhancements in beetroot seed productivity and quality have predominantly been investigated in sugar beet, while very scant data exist regarding table beetroot. In this respect, the application of growth stimulators and fertilizers to beetroot seed plants seems to be especially attractive due to their ability to improve phytohormone biosynthesis and seed nutrient storage [7,8,9]. Among growth stimulators, amino acid formulations [10], along with non-traditional Se [7,11] and Si derivatives [12,13], have drawn research interest.

Amino acids are the key compounds in seed development, providing energy and participating in seed storage protein and carbohydrate biosynthesis and secondary metabolite production [10,14]. They are known to be powerful immune and growth stimulators, regulating nitrogen homeostasis [15], cell division, and the production of IAA and GA phytohormones [10]. Various amino acid formulations include individual or complex mixtures of amino acids combined with humic acids and mineral fertilizers. Despite the individual differences in the beneficial effect of amino acids on seed production, their utilization is extremely valuable for improving seed yield and quality [15].

Among minerals, both Se and Si draw great research interest due to their ability to improve plants’ tolerance to environmental stresses, optimize plant nutrition, activate the biosynthesis of secondary metabolites, and increase plant yields and seed productivity [16]. In this respect, investigations on several plant species have revealed that the supply of Se at low concentrations both enhances seed yield and results in the formation of functional food products with high levels of this essential microelement for human organisms [7]. Among different Se derivatives, nanoparticles are the least toxic, demonstrating the highest ability to increase seed production in rice [17].

The biostimulant effect of silicon [18], its ability to activate phytohormone synthesis and enhance either plants’ resistance to environmental stresses [19] or the seed productivity of various plant species, represents the outstanding characteristics of this microelement. Previous research has indicated the highest efficiency of foliar treatment and the utilization of water-soluble ionic forms of Si [20,21].

The beneficial effect of soil yeast supply on the seed productivity of plants has been less intensively studied, despite its reported pathogen inhibition [22,23,24] and enhancement of nutrient bioavailability [25], phytohormone biosynthesis, and siderophore production [19,26]. Rhodotorula sp. F.C. Harrison, 1927 (Basidiomycota, Sporidiobolaceae) isolated from the Beta vulgaris L. (the Amarantaceae family) rhizosphere demonstrated plant growth-promoting properties via IAA biosynthesis, an increase in the solubility of phosphorous, and exopolysaccharide synthesis under stress conditions [27]. In previous research [28], the foliar supply of yeast extracts encouraged the accumulation of biologically active compounds stimulating plant growth, activating the synthesis of chlorophyll, proteins, and nucleic acids.

The present research study aimed to evaluate the effects of different plant biostimulants based on amino acids, nano-Se, Si, the soil yeast R. glutinis, and mineral fertilizer on the yield and quality of beetroot seeds and the peculiarities of seed formation, as well as the nutritional value and yield of the microgreens grown from these seeds.

2. Materials and Methods

2.1. The Growing Conditions and Experimental Protocol

This research study was carried out on beetroot (B. vulgaris L., cultivar Marusia; selection of Federal Scientific Vegetable Center—FSVC). The plants were grown at the FSVC experimental fields in the Moscow region, Russia (55°39.51′ N, 37°12.23′ E), in 2022–2023. The trial was conducted in a loam sod podzolic soil with the following characteristics: a pH of 6.2, 1.32 mg-eq 100 g⁻¹ hydrolytic acidity, 2.1% organic matter, a sum of absorbed bases of 93.6%, 18.5 mg kg⁻¹ mineral nitrogen, 21.3 mg kg⁻¹ ammonium nitrogen, 402 mg kg⁻¹ mobile phosphorous, and 198 mg kg⁻¹ exchangeable potassium. The mean temperature and precipitation during the crop cycle were measured using an Automated Weather Station AW310 (Vaisala Oyj, Vantaa, Finland) at the Federal Scientific Vegetable Center and are presented in

Table 1. Monthly temperature and precipitation in 2022–2023, during beetroot seed production (FSVC, Moscow region).

Month	Temperature (°C)		Precipitation (mm)
	2022	2023	2022	2023
May	10.0	12.2	55.5	33.9
June	18.6	16.5	24.6	66.9
July	20.2	18.1	66.1	83.9
August	22.3	19.9	13.7	48.8
September	9.6	14.9	125.7	10.2

.

The transplant of seed plants was practiced on 25 April, with a 0.33 m spacing between plants along the rows which were 0.50 m apart. Fertilization was performed by supplying 140 kg ha⁻¹ N, 60 kg ha⁻¹ P₂O₅, and 190 kg ha⁻¹ K₂O.

The experimental protocol was based on the comparison between the following six treatments plus an untreated control: (1) Aminosil, exclusively containing amino acids [29]; (2) Multimolig M, with a complex composition of amino acids, humic acids, and macro- and microelements; (3) Rhodotorula glutinis, a new soil yeast preparation with immunoregulating properties due to the presence of polysaccharides and amino acids [27,30]; (4) the silicon formulation ‘Siliplant’, containing potassium silicate and a small amount of macro- and microelements; (5) nano-Se [31]; (6) the mineral fertilizer Wuxal Macromix. A randomized complete block design with three replicates was arranged for the treatment distribution in the field. The experimental unit had a 20 m² (4 × 5 m) surface area.

The biostimulant characteristics and doses used are presented in

Table 2. Plant biostimulants and mineral element formulation applied to the aerial parts of beetroot plants during seed production in three phases of development.

Formulation	Dose	Composition	Company
Wuxal Macromix	2 mL L−1	A suspension fertilizer containing N: 240 g L−1; P: 240 g L−1; K: 180 g L−1; B: 0.3 g L−1; Cu: 0.75 g L−1; Zn: 0.75 g L−1; Fe: 1.5 g L−1; Mn: 0.75 g L−1; Mo: 0.015 g L−1.	Aglukon GmbH & Co. KG, Düsseldorf, Germany
Multimolig M	4 mL L−1	Amino acids; humic acids: 4%; N total: 4%; N-NO3: 0.5%; P2O5: 0.5%; K2O: 1%; dry matter: 25%; organic matter: 30%; Ca: 0.2%; S: 1.5%; Na: 0.5%; Mg: 0.3%; Fe: 10%; Mn: 1.3%; Zn: 1%; Cu: 1.5%; Co: 0.4%; Mo: 1%; B: 0.06%.	Multimolig, St. Petersburg, Russia
Aminosil	20 mL L−1	Proteinogenic and non-proteinogenic amino acids; N: 1.8%; P: 1.8%; K: 1.8%; Mg: 0.21%; Fe: 0.02%; Ca: 0.55%; Mg: 0.21%; Zn: 0.001%; Mn: 0.002%.	Aminosil, Belgorod, Russia
Siliplant	3 mL L−1	Si: 7.5–7.8%; K: 1.0%; Fe: 0.30 g L−1; Mg: 0.10 g L−1; Cu: 0.07 g L−1; Zn: 0.08 g L−1; Mn: 0.30 g L−1; Mo: 0.06 g L−1; Co: 0.02 g L−1; B: 0.094 g L−1.	NEST M Inc., Moscow, Russia
Nano-Se	1.27 mM; 100 mg L−1	100 nm particles obtained by laser ablation.	Institute of Metallurgy and Material Science, Moscow, Russia
Rhodotorula glutinis	1 mL L−1	Soil yeast Rhodotorula glutinis formulation with cell titer of 1.5–2.5 MM KOE mL−1 (a source of amino acids, proteins, and phytohormones).	Federal Scientific Vegetable Center, Moscow, Russia

. The applied doses are recommended by the producers, while R. glutinis was supplied according to the indications of Sinichenko et al. [30].

To optimize the efficiency of the formulations tested (Table 2), the planned treatments to the seed plants were repeated thrice, i.e., at the beginning and in the middle of peduncle formation (14 June and 10 July, respectively) and at the beginning of flowering (20 July). All of the treatments were performed at 6.00 p.m. to avoid possible plant burns and applied at 300 L per ha by a Solo hand-held knapsack sprayer. The biostimulants were supplied to the aerial plants’ parts to prevent their possible interaction with soil components, thus resulting in the highest absorption efficiency.

After harvesting on 20 September, the seed branches were immediately transferred to a flour dryer and dried under an intensive air flow at 20–25 °C for 1–2 weeks. The stems were separated, and the seeds were cleaned from dust and impurities by a Hoffman MFG seed blower (Hoffman Manufacturing Inc., Fogelsville, PA, USA) and then separated into four diameter classes (3.00–3.49; 3.50–3.99; 4.00–4.49; 4.50–4.99 mm) using Petkus MP 80 seed sieves (Pretkus Technologie GmbH, Wutha-Farnroda, Germany). The mentioned partitioning allowed to determine the peculiarities of seed formation under biostimulant supply. The mean weight of 1000 seeds within each seed class was measured by a seed counter, Pfeuffer C.E. (Pfeuffer GmbH, Kitzingen, Germany), and laboratory scales, AND GX-1000 (A2D Company Ltd., Abingdon, UK).

The following parameters of seed quality were assessed: germination energy (SGE) and germination capacity (SGC), yield, mean weight, and biochemical characteristics within each seed diameter class, along with the corresponding characteristics of microgreens associated to the two extreme seed size ranges.

2.2. Preparation and Characterization of Selenium Colloidal Solution

Nano-selenium colloidal solution was prepared at the Baikov Institute of Metallurgy and Material Science from pure Se pellets placed in distilled water by using the pulse laser ablation method via nanosecond Nd:YAG laser irradiation with a 1064 nm wavelength, and 12 ns and 2.5 J pulse duration and energy, respectively, concentrated on the target by a lens.

After drying at 50 °C, the selenium nanoparticles were analyzed by X-ray diffraction on an X-ray diffractometer, Shimadzu XRD-600, in the 2θ range from 10° to 55° at a tube voltage of 40 kV and a current of 100 mA. To identify the diffraction peaks, a comparison of the results with literature data and the ICDD database (International Centre for Diffraction Data Power Diffraction File; 2 Campus Blvd: Newtown Square, PA, USA, 2007) was carried out.

The typical diffraction peaks of 23.7, 29.8, 41.3, 43.7, 45.5, and 51.7 corresponded to the crystal planes of crystalline Se (100, 101, 110, 102, 111, and 201).

Dynamic Light Scattering (DLS) analysis on a Photocor Compact Z (Photocor, Beltsville, MD, USA) laser analyzer with a λ = 589 nm wavelength and a laser rated-power output of 32 mW at 25 °C revealed a narrow nanoparticle size distribution with a 90 nm average size (Figure 1). The ζ-potential of the nano-Se colloidal solution (−36.2 mV) entails repulsion of nanoparticles hampering aggregation.

To evaluate the Se nanoparticle concentration, ICP-AES spectrometry was used (ULTIMA 2 spectrometer; Horiba Jobin Yvon, Palaiseau, France). The selenium content was about 17 mg L⁻¹.

2.3. SGE and SGC Determination

Four replicates (one hundred pure seeds per replicate) of each seed class (3.00–3.49 mm; 3.50–3.99 mm; 4.00–4.49 mm; 4.50–4.99 mm), associated to all of the experimental treatments applied, were germinated on a blotter in plastic bags at 25 °C in the dark at a pH of 7.2 [32]. To ensure the relative stability of the concentrations used, water was supplemented once every 24 h. Seed germination energy (SGE) was determined on the 5th day and calculated in % of sprouted seeds with respect to the total seed number, while seed germination capacity (SGC) was measured analogically on the 10th day of germination using the following equations:

SGC (%) = (Total germinated seed after 10 days):(Total seed sown) × 100

SGE (%) = (Germinated seeds after 5 days):(Total seed sown) × 100

2.4. Microgreen Preparation

To disinfect the seeds for microgreen production and decrease the growth inhibitor content, seeds of two opposite size classes (4.50–4.99 mm and 3.00–3.49 mm diameter) were soaked in water for 1 h using a light pink solution of permanganate. Then, the water excess at the seed surface was removed with filter paper, and the seeds were sown in 5 × 5 × 4 cm pots containing a peat and vermiculite mixture (5:1), with a 3–4 mm distance between seeds (about 200 seeds m⁻²). The soil was pressed to enhance the contact with seeds and sprayed with water, and the pots were covered with polyethylene film and kept at 20 °C for 3 days. After removing the polyethylene film, the seedlings and microgreens were watered for 14 days up to the formation of the first two true leaves. The experiment was conducted in triplicate. The microgreens were harvested 14 days after germination, and their fresh weight (FW) was measured. The chlorophyll and betalain contents were determined on fresh homogenates, while other parameters were assessed on dry homogenized samples after drying microgreens at 70 °C to constant weight.

2.5. Selenium

The selenium content in seeds and microgreens was measured using the micro-fluorimetric method [33] based on the acidic digestion of dry homogenized seeds/microgreens’ powder with a mixture of nitric and perchloric acids, the subsequent conversion of selenate (Se⁺⁶) into selenite (Se⁺⁴) using a solution of 6 N HCl, and the fluorescence value determination of piazoselenol, formed as a result of the condensation between Se⁺⁴ and 2,3-diaminonaphtalene. The analysis was performed in hexane at 519 nm λ emission and −376 nm λ excitation. As an external standard, Se-fortified mitsuba stem powder with a Se content of 1865 µg kg⁻¹ (Federal Scientific Vegetable Center) was used. The results are expressed in µg kg⁻¹ d.w., as means of three replications.

2.6. Chlorophyll and Carotene Determination

A spectrophotometric analysis was used for the determination of chlorophyll and carotene in beetroot microgreen ethanolic extract, using the values of the light absorption at 470 nm, 646 nm, and 664 nm through a spectrophotometer (Unico 2804 UV, Suite E Dayton, NJ, USA), according to the following equations [34]:

chl a = 13.36A₆₆₄ − 5.19A₆₄₉;

chl b = 27.43A₆₄₉ − 8.12A₆₆₄;

carotene = (1000A₄₇₀ − 2.13 chl a − 87.63 chl b): 209;

where A is the light absorbance, chl a is chlorophyll a, and chl b is chlorophyll b.

The results represent the means of three determinations and are expressed in mg per 100 g of dry leaf weight.

2.7. Betalain Pigment Determination

Betaxantin (BX) and betacianin (BC) in beetroot microgreens were determined spectrophotometrically by the light absorption levels of water extracts at 535 nm and 485 nm, using the following equations [35]:

BC = (D₅₄₃ × V × 500 × d):(60,000 × m)

BX = (D₄₈₅ × V × 308 × d):(48,000 × m)

where D₅₄₃ and D₄₈₅ are the light absorption levels at 543 and 485 nm, respectively; 500 is the molecular weight of betacyanin; 308 is the molecular weight of betaxantin; 60,000 and 48,000 are the extinction levels of betacyanin and betaxanthin, respectively; m is the sample weight in g; and d is the dilution.

The results represent the means of three determinations and are expressed in mg per 100 g of dry leaf weight.

2.8. Total Polyphenols (TP)

Half a gram of dry homogenized seeds/microgreens’ powder was extracted with 20 mL of 70% ethanol for 1 h at 80 °C [35]. The mixture was cooled down and quantitatively transferred to a volumetric flask, the volume was adjusted to 25 mL, and the mixture was filtered through filter paper. A volume of 1 mL of the resulting solution was transferred to a 25 mL volumetric flask and mixed with 2.5 mL of saturated Na₂CO₃ solution and 0.25 mL of diluted (1:1) Folin–Ciocalteu reagent. After adjusting the volume to 25 mL with distilled water, the reaction mixture was left at room temperature for an hour. The solutions obtained were analyzed with a spectrophotometer (Unico 2804 UV, Suite E Dayton, NJ, USA) by evaluating the absorption value at 730 nm. As an external standard, 0.02% gallic acid was used. The results are expressed as mg of Gallic Acid Equivalent per g of dry weight (mg GAE g⁻¹ d.w.).

2.9. Antioxidant Activity (AOA)

The antioxidant activity was assessed via the titration of 0.01 N KMnO₄ solution with ethanolic extracts of dry samples [35]. The results are expressed in mg Gallic Acid Equivalent per g of dry weight (mg GAE g⁻¹ d.w.).

2.10. Proline

Proline concentration was determined according to Ábrahám et al. [36], with slight modifications. About 50 mg of dry homogenized plant seeds were ground with 10 mL of 3% sulfur salicylic acid in a mortar. The mixture was filtered, and 1 mL of the resulting filtrate, 2 mL of ninhydrin reagent, and 2 mL of acetic acid were incubated in tubes at 95 °C for 1 h. After cooling the tubes, the samples were extracted with 3 mL of toluene, and the proline concentration was measured by using the absorption value of the toluene extract at 520 nm and a calibration curve with 5 different proline (Merck, Rahway, NJ, USA) concentrations (0–40 µg mL⁻¹).

2.11. Statistical Analysis

All of the values are the means of three replicates per experimental treatment. The data were processed using both one-way and two-way analyses of variance, and mean separations were performed with Duncan’s multiple range test, referring to the 0.05 probability level, by a SPSS software version 29 (Armonk, NY, USA). Correlations between the examined parameters were carried out the using Student’s test.

3. Results and Discussion

3.1. Seed Yield

The treatment of seed plants with biostimulants significantly affected the beetroot seed yield (Figure 2), with the Siliplant and nano-Se formulations resulting in the highest production; all the applications were more effective than the untreated control, except R. glutinis, which also did not statistically differ from Multimolig M, Aminosil, and Wuxal Macromix, both in 2022 and 2023 (Figure 2). The non-significant effect of the research year on the examined variables indicates the stability of the beneficial influence of the applied biostimulants.

Though Si is known to be a good growth stimulator, both at high and low concentrations for various agricultural crops [21,37,38], its effect on seed yield has been studied in few crops, among which rice, maize, and soybean [37,39]. The detected phenomenon of 18.2–20.2% seed production increase upon the Si supply may relate to the enhancement in plant nutrient accumulation and phytohormone biosynthesis [19,37].

The Nano-Se showed a similar beneficial effect on seed yield to that recorded for Si, increasing seed yield by 15.2–16.0%. Indeed, Se is a well-known growth stimulator, immune modulator, and natural antioxidant capable of modulating gene expression and secondary metabolite biosynthesis [31]. In our research, unlike Siliplant, Multimolig M, Aminosil, and Wuxal Macromix, the nano-Se formulation did not provide the plants with even small additional nutrients as the mentioned formulations, thus confirming that its high beneficial effect exclusively derived from nanoparticles. Among the different forms of Se, i.e., selenate, selenite, organic, and nanoparticles, the latter have displayed the lowest toxicity, which is a favorable characteristic for plants. In rice, among the different Se forms supplied, nanoparticles led to higher grain yield compared with Se organic and inorganic compounds [17]. Nevertheless, the species variability of the plant response to Se supply indicates a need for further studies to unveil the optimal Se form in seed beetroot plant treatment.

The amino acid application (Aminosil and Multimolig M) to seed beetroot plants was not so effective as the Se and Si ones, increasing seed yield by less than 6.7%, though amino acids are known to activate the synthesis of several enzymes, enhancing antioxidant defense and osmoregulation, affecting gene expression, and increasing nutrient availability [40,41] and plant seed productivity [40,42]. Being also a natural source of amino acids for plants, the soil yeast formulation (R. glutinis) did not increase beetroot seed yield, compared with the untreated control, showing only a tendency to increase seed production.

The mean values of 1000 seed weight associated with the four seed size classes were in the 7.3–20.9 g range (

Table 3. The mean weight of beetroot seeds (g per 1000 seeds) within each diameter class for control plants and plants treated with biostimulants.

Parameter	Seed Diameter Class (mm)
	3.00–3.49	3.50–3.99	4.00–4.49	4.50–4.99
Untreated Control	7.55	10.24	14.5	21.71
Wuxal Macromix	6.96	9.88	14.8	19.69
Multimolig M	7.56	10.35	15.0	20.44
Aminosil	6.88	10.13	14.9	20.76
Siliplant	6.97	9.87	15.5	21.10
Nano-Se	7.41	9.85	15.2	21.50
R. glutinis	7.52	10.47	15.4	20.92
Significance	n.s.	n.s.	n.s.	n.s.
M ± SD	7.26 ± 0.31 d	10.11 ± 0.25 c	15.00 ± 0.35 b	20.87 ± 0.67 a
CV (%)	4.29	2.47	2.53	3.24

CV: coefficient of variation; n.s.: not significant; different letters for mean values refer to significant differences between the values according to Duncan’s test at p < 0.05. M ± SD: mean ± standard deviation.

). Our results indicate that the variations in seed mean weight were not significant within each seed diameter class under different biostimulant treatments, with CV values not exceeding 4.29%.

Considering that beetroot seeds with a diameter greater than 4.0–4.5 mm provide a significantly higher marketable yield of roots [43], we investigated the seed size class distribution of seed plants treated with the abovementioned biostimulants and fertilizers.

3.2. Seed Size Class Distribution

The mean diameter class distribution of beetroot cv. Marusia seeds for control plants in the two-year trial (Figure 3) was the following: 19.7% (3.00–3.49 mm), 10.4% (3.50–3.99 mm), 24.4% (4.00–4.49 mm), and 44.5% (4.50–4.99 mm); it significantly differs from the corresponding data previously published for cv. Bordo [43]. Indeed, the two-year data related to the seed diameter class distribution of cv. Bordo [43] revealed a higher percentage of the large seed range (4.50–4.99 mm; 60.5%) and a lower occurrence of the small seed class (3.00–3.99 mm; 10.5%), suggesting that seed size is genetically determined, which is in agreement with previous reports on sugar beet [44].

The treatment of beetroot seed plants with biostimulants and fertilizers, both in 2022 and 2023, did not significantly change the general pattern of seed diameter class distribution: (4.50–4.99 mm) > (4.00–4.49 mm) > (3.00–3.49 mm) > (3.50–3.99 mm) (Figure 3). On the contrary, R, glutinis supply led to an unusual, similar distribution of all of the seed size classes (23–26%), while Si application resulted in the highest occurrence of seeds belonging to the two greatest diameter classes (4.00–4.99 mm; 81.4%) and the lowest percentage of 3.00–3.49 mm size seeds, compared with the other formulations applied. Besides the improvement in plant nutrition, the observed beneficial effect of silicon may be explained by its participation in regulating the biosynthesis of phytohormones [45], which directly affect the seed development and size [46]. The effectiveness of Si and Se on the seed yield (Figure 2) is in accordance with the close relationship between Se and Si, and phytohormones [47].

The phenomenon of the unusual, similar distribution of all of the seed size classes for plants treated with R. glutinis entails the existence of significant changes in seed morphology caused by soil yeast supply (Figure 3). In this respect, it should be considered that the biological effect of the foliar yeast treatment mostly reflects the presence of secondary derivatives of yeast metabolism, such as amino acids, polyphenols, etc. [28]. However, further investigations are needed to unveil the factors involved in seed size changes due to R. glutinis supply, which should also involve the chemical composition of yeast secondary metabolites and their concentration effect.

3.3. Seed Germination Energy and Capacity

From the significant interactions between the plant biostimulant treatment and the seed size class, it arose that seed germination energy (SGE) and capacity (SGC) continuously increased with the rise in seed size regardless of the treatment (

Table 4. Interaction between plant biostimulant treatment and seed size class on beetroot total seed germination energy (SGE) in % in 2022 and 2023.

Treatment	Seed Diameter Class (mm)
	3.00–3.49		3.50–3.99		4.00–4.49		4.50–4.99
	2022	2023	2022	2023	2022	2023	2022	2023
Control	36 c	35 c	53 b	54 b	70 ab	69 b	94 a	95 a
Wuxal Macromix	47 b	48 b	70 a	71 a	80 ab	79 ab	97 a	97 a
Multimolig M	35 c	34 c	65 a	64 ab	77 ab	78 ab	96 a	97 a
Aminosil	72 a	73 a	75 a	74 a	85 a	86 a	97 a	98 a
Siliplant	46 b	45 b	66 a	67 ab	75 ab	76 ab	94 a	95 a
Nano-Se	67 a	66 a	72 a	73 a	80 ab	81 a	94 a	95 a
R. glutinis	44 b	43 b	42 c	41 c	66 b	65 b	84 a	83 a

Values with the same letter do not differ statistically according to Duncan’s test at p < 0.05.

and

Table 5. Interaction between plant biostimulant treatment and seed size class on seed germination capacity (SGC) in % in 2022 and 2023.

Treatment	Seed Diameter Class (mm)
	3.00−3.49		3.50−3.99		4.00−4.49		4.50−4.99
	2022	2023	2022	2023	2022	2023	2022	2023
Control	44 c	43 c	60 bc	61 bc	76 ab	75 ab	95 a	95 a
Wuxal Macromix	55 b	54 b	77 a	76 a	80 ab	81 a	97 a	97 a
Multimolig M	46 bc	45	80 a	79 a	87 a	88 a	97 a	98 a
Aminosil	80 a	81 a	82 a	83 a	87 a	86 a	97 a	98 a
Siliplant	52 bc	53 b	72 ab	73 a	80 ab	81 a	96 a	97 a
Nano-Se	76 a	75 a	82 a	83 a	87 a	88 a	96 a	97 a
R. glutinis	53 bc	52 b	54 c	53 c	70 b	71 b	87 a	86 a

Values with the same letters do not differ statistically according to Duncan’s test at p < 0.05.

).

According to the literature data, the great size and weight of beetroot seeds are characterized by high levels of germination energy and capacity [43]. The results of the present research are in accordance with the mentioned investigation and indicate the increase in the SGE and SGC values from small (3.00–3.49 mm) to large-sized (4.50–4.99 mm) seeds by 1.9 and 1.6 times, respectively (Table 4 and Table 5). On the other hand, the highest SGC/SGE variability in response to the experimental treatments corresponded to the seeds belonging to the 3.00–3.49 mm class (Figure 4). The latter outcome was confirmed by a significant CV value reduction with the increase in seed size both for SGE and SGC.

Indeed, the highest seed SGE and SGC values of 94–97% corresponded to the 4.50–4.99 mm class in all of the cases, except R. glutinis-treated seeds, which showed significantly lower values. The latter negative effect on beetroot seed germination contradicts the literature reports about the immunoregulating and growth-promoting properties of R. glutinis. Indeed, in previous findings [30], R. glutinis enhanced germination energy, plant growth, and resistance to abiotic stresses (temperature and humidity fluctuations), as well as the yield and quality of vegetables. The mentioned phenomenon presumably relates to the complexity of the R. glutinis effect connected with its ability to produce high levels of lipids, carotenoids, and exopolysaccharides [26,48].

The highest differences between the experimental treatments, corresponding to the 3.00–3.49 mm size class, are consistent with the beneficial effect on priming, showing a higher efficiency in sugar beet seeds with lower vigor [49]. Indeed, no positive effect of Multimolig M was recorded in the 3.00–3.49 mm seed diameter class, while the highest SGC increase was elicited by the nano-Se and Aminosil treatments (by 1.7–1.8 times, compared to the control). A much lower effect was observed with Siliplant, Wuxal Macromix, and R. glutinis supply (by 1.2–1.25 times).

Overall, the obtained results reveal the highest differences between the effects of the experimental treatments on the SGC/SGE values of the 3.00–3.49 mm seed size class, which dramatically decreased with the increase in seed weight/diameter and show negligible differences within the 4.50–4.99 mm class.

The beneficial effect of the Se application is in accordance with previous findings regarding the nano-Se effect on the seed germination capacity of various nanoparticle shapes and different plant species, predominantly cereals and legumes [50,51], with significantly less information for vegetables [7]. No data have been published so far regarding beetroot seed production both under Se and/or Si supply. The high levels of the seed SGC/SGE recorded under amino acid application are in accordance with the literature reports [40].

3.4. Antioxidant Status and Proline Content

The highest total antioxidant activity and polyphenol content were recorded in the smallest seed class (

Table 6. Interaction between plant biostimulant treatment and seed size class on beetroot seed antioxidant activity and polyphenol content in 2022 and 2023.

Treatment	AOA (mg GAE g−1 d.w.)				TP (mg GAE g−1 d.w.)
	3.00–3.49 mm		4.50–4.99 mm		3.00–3.49 mm		4.50–4.99 mm
	2022	2023	2022	2023	2022	2023	2022	2023
Control	22.3 ab	20.9 a	14.5 c	13.7 bc	9.5 a	8.3 a	7.5 a	6.7 a
Wuxal Macromix	19.2 b	16.8 b	13.9 c	12.9 c	8.0 a	6.8 b	5.7 b	4.9 b
Multimolig M	21.7 b	19.9 a	14.0 c	13.3 bc	8.1 a	7.5 ab	6.9 a	6.1 a
Aminosil	19.6 b	18.4 a	16.7 ab	15.6 ab	7.9 a	7.3 ab	6.9 a	5.7 ab
Siliplant	22.0 ab	21.2 a	17.8 ab	16.8 a	8.9 a	8.1 a	7.5 a	6.5 a
Nano-Se	23.5 ab	22.3 a	18.6 a	16.8 a	8.5 a	7.7 ab	6.8 ab	6.0 a
R. glutinis	26.0 a	22.4 a	15.8 bc	15.0 ab	8.8 a	7.4 ab	7.9 a	6.5 a

AOA: total antioxidant activity; TP: total phenolics; d.w.: dry weight. Values with the same letter do not differ statistically according to Duncan’s test at p < 0.05.

). Indeed, the total antioxidant activity (AOA) of the small-sized seeds exceeded that of the large ones by 1.38, while the corresponding value for polyphenol content was 1.25. Notably, biostimulant application did not significantly affect the total polyphenol content within the smallest seed class.

The results are in accordance with the observation by Simić et al. [52], who reported that under conditions of accelerated aging, the phenolic content in seed exudates negatively correlated with germination. Furthermore, the higher levels of polyphenol content and total antioxidant activity in the small seeds suggest the existence of a more intensive oxidative stress compared with large-sized seeds. The latter statement is confirmed by the twice-higher proline concentration in the small-sized seeds compared with the 4.50–4.99 mm ones (Figure 5).

Considering that the proline concentration reflects the stress level caused by different environmental factors [53], the results indicate the existence of a more significant oxidative stress in the small-sized seeds, which is in accordance with the significantly higher levels of AOA and TP in the same seeds. The decrease in proline content in beetroot seeds was detected due to the application of the biostimulants and fertilizers tested, except R. glutinis. Notably, all of the mentioned treatments showed much higher effects on the small seeds, exposed to a more intensive oxidative stress, compared with the large-sized seed class, which was not significantly affected by the applied experimental factors (Figure 5). In this respect, the decrease in oxidative stress in the small-sized seeds was most pronounced under Aminosil and Siliplant supply, which indicates the suitability of the mentioned biostimulants for enhancing the antioxidant defense in beetroot seeds belonging to both the small- and large-sized classes.

As can be observed in Table 6 and Figure 5, the R. glutinis treatment was the only exception, resulting in a significant proline increase in the small seeds and a decrease in the large-sized class ones. Further investigation will be useful to reveal the mechanism of R. glutinis effect on seeds.

3.5. Microgreen Yield

Despite the significant differences in antioxidant defense and proline content between the small- and large-sized seeds, both of them showed a similar ability to produce a high yield of microgreens (

Table 7. Interaction between plant biostimulant treatment and seed size class on length and yield of beetroot microgreens.

Treatment	Microgreen Length (cm)		Microgreen Yield (g per 100 Microgreens)
	3.00–3.49 mm	4.50–4.99 mm	3.00–3.49 mm	4.50–4.99 mm
Control	11.5 d	11.9 d	94 c	99 c
Wuxal Macromix	11.6 d	12.5 cd	100 c	108 bc
Multimolig M	11.9 d	14.3 bcd	118 b	124 b
Aminosil	14.5 bc	15.2 ab	131 ab	136 ab
Siliplant	14.8 abc	15.7 ab	135 ab	139 ab
Nano-Se	15.3 ab	17.8 a	137 ab	154 a
R. glutinis	12.8 cd	14.1 bcd	127 ab	130 ab
M ± SD	13.2 ± 1.6	14.5 ± 2.0	120.3 ± 17.1	127.1 ± 18.8
CV (%)	12.1	11.8	14.2	14.8

For each parameter and within each column, values with the same letter do not differ statistically according to Duncan’s test at p < 0.05; M ± SD: mean ± standard deviation; CV: coefficient of variation.

). In this respect, the biostimulant application to seed plants was the pivotal factor for the subsequent production of microgreens with Siliplant, nano-Se, and Aminosil, which had the highest beneficial effect (Table 7, Figure 6). Indeed, the yield increase ranged from 39.4 to 47.9% for the 3.00–3.49 mm seed size class and from 36.4 to 55.6% for the 4.50–4.99 mm diameter class, which displayed the highest percentage under the nano-Se treatment. The amino acids–humic acids-based biostimulant Multimolig M and the mineral fertilizer Wuxal Macromix were less effective than the other biostimulants (Table 2, Figure 6). Notably, no significant differences were recorded between the small- and large-sized seed classes both in terms of length and yield of the microgreens produced (Figure 6).

Interestingly, despite its relatively low effect on seed yield and quality, the R. glutinis formulation showed a growth stimulation of microgreens, increasing the production by 31.3–35.1%. According to the literature reports, beetroot microgreen yield is affected by genetic peculiarities, seed density, nutrient supply, moisture availability, and the utilization of artificial (red) light [54]. Microgreens are characterized by a significantly higher antioxidant status [4,5] and the bioavailability of biologically active compounds [6] beneficial to human health [55,56], compared to adult plants. Furthermore, beetroot microgreens showed significantly higher antioxidant levels compared to the microgreens of other vegetables (radish, daikon, pea, and onion) [55], suggesting the special importance of seed quality. The results of the present research study indicate that at the appropriate seed density (200 g m⁻²) [57], the microgreen yield may be increased using the seeds of plants treated with Si, nano-Se, or amino acids.

The beneficial Se effect recorded in the present study agrees with previous investigations on beetroot Se biofortification, resulting in a significant increase in betalain content [58]. On the contrary, no up to date information is available about the effect of Si, amino acids, and soil yeast formulations on betalain pigment accumulation in beetroot and its microgreens.

The obtained results indicate that the treatments applied to the seed plants did not significantly affect the total antioxidant activity and polyphenol (TP) content in microgreens (

Table 8. Interaction between plant biostimulant treatment and seed size class on antioxidant status of beetroot microgreens.

Treatment	Betalain Pigments		Total chl		Carotene		AOA		TP
	mg 100 g−1 d.w.						mg GAE g−1 d.w.
	3.00– 3.49 mm	4.50– 4.99 mm	3.00– 3.49 mm	4.50– 4.99 mm	3.09– 3.49 mm	4.50– 4.99 mm	3.00– 3.49 mm	4.50– 4.99 mm	3.00– 3.49 mm	4.50– 4.99 mm
Control	5.8 b	6.3 b	88 c	89 e	8 d	9 c	42.1	41.1	16.1	14.9
Wuxal Macromix	7.1 a	7.6 a	90 c	112 d	9 cd	10 c	42.1	40.8	17.0	15.0
Multimolig M	7.0 a	7.6 a	91 c	121 c	10 c	9 c	45.6	43.9	19.0	18.0
Aminosil	7.3 a	7.8 a	123 a	134 b	13 a	13 b	43.4	42.8	18.1	16.6
Siliplant	7.7 a	7.7 a	126 a	157 a	14 a	17 a	46.7	45.9	19.2	16.4
Nano-Se	7.6 a	7.8 a	127 a	151 a	13 a	18 a	50.0	47.4	18.4	17.7
R. glutinis	6.9 a	7.6 a	108 b	130 b	12 b	13 b	46.0	40.5	17.7	15.9
Significance							n.s.	n.s.	n.s.	n.s.
M	7.1 a	7.5 a	107.6 b	127.7 a	11.3 a	12.7 a	45.1 a	43.2 a	17.9 a	16.4 a
SD	0.6	0.5	17.9	23.2	2.3	3.7	2.8	2.7	1.1	1.2
CV (%)	8.5	6.7	16.6	18.2	20.4	29.1	6.2	6.3	6.1	7.3

AOA: total antioxidant activity; TP: total phenolic content; chl: chlorophyll; n.s.: not significant. Values with the same letter do not significantly differ according to Duncan’s test at p < 0.05; M: mean; SD: standard deviation; CV: coefficient of variation.

).

The data presented in Table 8 suggest that all of the treatments fostered the betalain pigment biosynthesis, with relatively low differences in pigment accumulation among the experimental treatments. On the contrary, higher differences in photosynthetic pigment levels were recorded among the various microgreens produced, with CV values of 16.6–18.2% for chlorophyll and 20.4–29.1% for carotene (Table 8), indicating a beneficial effect of Siliplant, nano-Se, and Aminosil on the mentioned seedling growth. The latter outcome was confirmed by the positive correlations between microgreen yield/length and chlorophyll/carotene content (r = 0.889 and 0.937, at p < 0.001), reflecting the activation of photosynthesis, the enhancement in photo-pigment accumulation and the efficiency of light energy transmission, especially significant for nano-Se and Siliplant supply (

Table 9. Correlation coefficients for the examined parameters of microgreens (n = 14).

	MG Carotene	MG Yield	MG Length	MG BP	Seed Pro	Seed AOA	SGC
MG Chl	0.899 a	0.892 a	0.937 a	0.797 a	−0.550 d	−0.133	0.587 c
	MG Carotene	0.899 a	0.889 a	0.656 e	−0.361	0.124	0.347
		MG yield	0.934 a	0.780 a	−0.396	0.146	0.352
		MG length		0.746 a	−0.537 f	−0.036	0.524 f
				MG BP	−0.677 e	−0.241	0.797 a
					Seed Pro	0.598 c	−0.637 b
						Seed AOA	−0.781 a

Chl: chlorophyll; MG: microgreens; BP: betalain pigments; Pro: proline; AOA: total antioxidant activity; SGC: seed germination capacity. (a) p < 0.001; (b) p < 0.01; (c) p < 0.02; (d) p < 0.03; (e) p < 0.005; (f) p < 0.05.

).

The different experimental treatments did not significantly affect the betacyanin/betaxanthin ratio (1.17 in the chosen cultivar), independently on the seed size class. Microgreen yield and length were positively correlated to the intensity of photosynthetic and betalain pigment biosynthesis (r = 0.747 and 0.780, at p < 0.001; Table 9). A significant correlation was also recorded between betalain accumulation in the microgreens and seed germination capacity, which indicates a positive correlation between microgreen yield and quality, and seed viability. On the other hand, the variations in the seed proline values (Table 6) are consistent with its significant negative correlation with SGC and betalain pigment levels in the microgreens.

Additionally, it is worth highlighting that despite a narrow safety concentration range for Se consumption by the human organism, no risks of Se overdose with beetroot microgreen utilization exist. Indeed, the Se levels in the beetroot seeds were 466.3 ± 16.8 µg kg⁻¹ d.w. under nano-Se supply, regardless of the seed size class, and exceeded the corresponding value in the control seeds by 18.5 times. The microgreens produced from the mentioned seeds demonstrated an even lower Se content, i.e., 311 µg kg⁻¹ d.w. Considering the recommended daily allowance (RDA) value of 70 µg day⁻¹ for Se consumption [59], 100 g of Se fortified microgreens will provide about 3 µg Se. In this respect, further investigations are needed to reveal the possibility of microgreen Se level increase either via an increase in the nano-Se dose or by applying more bioavailable forms of Se, such as selenates [60] or organic derivatives [31].

4. Conclusions

The results of the present investigation indicate a significant increase in beetroot seed yield and viability under Si and nano-Se supply, leading to enhanced production and quality of the microgreens grown from the mentioned seeds. All of the biostimulants applied, except R. glutinis, elicited a decrease in oxidative stress in the seeds, especially within the small-sized class, with the highest beneficial effect of the Aminosil and Siliplant treatments. The high seed germination capacity and energy of the large-sized seed classes, regardless of the biostimulant supply, contrary to the small-diameter class, suggest a more powerful effect of the biostimulants on the quality of small seeds. Despite the stability of the small- and large-sized seed distribution within the total seed lot, only the application of Si led to an increase in the large-sized seed range, while R. glutinis fostered a homogenous distribution of seeds among the different diameter classes. The recorded beneficial effect of the biostimulants on beetroot seed plants provides the basis for enhancing the seed production and quality of this species, thus meeting the requirements of the Sustainable Development Goals of the United Nations aimed at fighting hunger and improving human health and well-being.