The Influence of Nitrogen and Sulfur Fertilization on Oil Quality and Seed Meal in Different Genotypes of Winter Oilseed Rape (Brassica napus L.)

Abstract

Adequate nitrogen (N) and sulfur (S) fertilization of oilseed rape crops is necessary to obtain good-quality oil and post-extraction rapeseed meal. The aim of this study was to determine the effect of different doses of N fertilization (100, 160 and 220 kg ha⁻¹) and S (0, 30, 60 and 90 kg ha⁻¹) on the value of seeds of three winter oilseed rape genotypes. Two winter oilseed rape genotypes obtained by mutagenesis (cultivar Polka and breeding genotype PN440) were characterized by changed fatty acid profile. The cultivar Polka, type HO (high oleic), had a high content of oleic acid (C18:1, 78.0%) and the breeding genotype PN440, type HOLL (high-oleic and low-linolenic), had a high content of oleic acid (C18:1, 75.0%) and a low content of linolenic acid (C18:3, 3.0%). We also used the canola type of winter oilseed rape cultivar, Monolit. The analysed winter oilseed rape genotypes responded similarly to the N and S fertilization factors with regard to the content of crude fat and total protein in the seeds and the composition of fatty acids in the oil. N fertilization increased the content of glucosinolates (GLS-alkenyl, indole and total) in seeds, whereas S application decreased the content of saturated fatty acids (stearic acid-C18:0) in oil and increased the content of alkenyl and total glucosinolates (GLSs) in seeds. A significant interaction between N and S was observed for crude-fat and total-protein content. This study suggests that ensuring an adequate supply of both nitrogen and sulfur in the soil is essential for optimizing meal and oil quality in different types of winter oilseed rape cultivars. Proper management of these nutrients can lead to improved oil content and overall crop performance.

1. Introduction

Rapeseed (Brassica napus L.) is one of the world’s most important oilseed crops. The growing demand for a healthy source of oil for human nutrition, biofuel production and rapeseed meal as a high-quality animal feed has led to a significant increase in the global rapeseed production to over 85 million tonnes in 2023, of which 3.6 million tonnes are in Poland [1].

Oilseed rape has global acceptance, largely due to significant advances in the quality of its seed oil, characterized by the absence of erucic acid (C22:1), and seed meal with a very low glucosinolate content [2,3].

The value of rapeseed oil for food and technical applications is determined by its fatty acid composition. The oil obtained from the seeds of currently cultivated winter oilseed rape varieties contains approximately 62% oleic acid (C18:1), 20% linoleic acid (C18:2), 10% linolenic acid (C18:3) and 1% eicosenoic acid (C20:1), and the remaining 7% is composed of saturated fatty acids (SFAs) such as palmitic acid (C16) and stearic acid (C18:0) [4]. Due to the high content of C18 unsaturated fatty acids (EFAs), especially polyunsaturated fatty acids (PUFAs) such as linoleic acid (C18:2, ω-6) and linoleic acid (C18:3, ω-3), winter oilseed rape oil is considered one of the healthiest vegetable oils [5,6]. Oils high in polyunsaturated fatty acids (PUFAs) (about 30%) have low oxidative stability and are therefore undesirable for food (e.g., deep frying) and biofuel production, because such oils cannot be stored for long periods. To overcome this problem, chemical mutagenesis and plant selection have been used to obtain oilseed rape genotypes that produce oil with a high content of oleic acid (C18:1) (over 75%; HO type) and with a high content of oleic acid (C18:1) and a low content of linolenic acid (C18:3) (about 75% and less than 3%, respectively; HOLL type) [4,7].

The chemical composition of rapeseed is mainly determined by genotype (cultivar). However, it can also be regulated by agrotechnical (fertilization, harvesting date and method) and environmental factors (temperature, water availability, and length of the growing season) [8,9,10,11]. Among the agrotechnical treatments, the crude fat and protein content of oilseed rape is mainly influenced by N fertilization [12]. Nitrogen reduces the crude fat content and increases the total protein content of Brassica seed [13,14,15]. This, in turn, causes significant changes in the fatty acid composition [14] and also affects the biosynthesis of GLS [13,14,15,16,17]. Sulfur is an essential nutrient for the proper growth and development of plants, as well as for increasing production and obtaining good-quality crops. Due to its role in various metabolic functions in plants (in the synthesis of proteins, carbohydrates, lipids and chlorophyll, and in photosynthesis), it has a significant effect on the amount of protein and oil accumulated in seeds [8,18,19,20] and modifies the fatty acid profile [21].

Brassicaceae oilseeds have a high requirement for S. In particular, winter oilseed rape has the highest requirement (15–20 kg S Mg⁻¹ seed) for this component [22]. The need for S fertilization may be related to the progressive depletion of this element from soils due to reduced dry- and wet-S precipitation from the atmosphere and the increasing production of Brassica crops in agricultural ecosystems [23,24]. It has been estimated that in Poland the annual dry- and wet-S precipitation from the atmosphere to the soil surface decreased from 51 kg ha⁻¹ in 1990 to 9 kg ha⁻¹ in 2017 [25]. In most areas of Poland, the amount of sulfate sulfur (S-SO₄) available to plants from the soil does not exceed 20 mg S-SO₄ kg⁻¹, and more than half of the agricultural soils in the country have a low S-SO₄ content (less than 10 mg S-SO₄ kg⁻¹) [26]. Studies have shown a variable effect of S fertilization on the fat content of winter oilseed rape. The increase in fat content as a result of S fertilization has been reported by several authors [27,28], especially under conditions of S deficiency [20]. On the other hand, a decrease in seed fat content has been observed in sites with sufficient S supply [29,30,31], or no clear effect of S fertilization on this parameter has been observed [23,32].

S fertilization may also affect the nutritional value of oil and non-fat seed residues [33]. Studies on winter- and spring-rape cultivars have shown an increase in fat content and a decrease in oleic (C18:1) acid content in response to S fertilization [8,34]. Ahmad and Abdin [35] and Barczak [36] observed an increase in oleic (C18:1) acid content in mustard oil. Based on their observations, Ahmad and Abdin [37] suggested that the interaction of N and S affects not only the quantity but also the quality of rapeseed and mustard oils. The authors found that the content of oleic (C18:1) and linoleic (C18:2) acids increased when S and N were used in combination.

Both N and S play an important role in protein synthesis in plants. Insufficient S negatively affects the synthesis of S-amino acids (mainly methionine and cysteine) and therefore reduces the amount and quality of protein in plants [33,38]. A balanced supply of N and S allows the production of oil with optimum quality [39,40] and protein content [41]. The positive effect of S fertilization on protein content in oilseed rape has been reported by many authors [14,18,20,28,31,32]. Sulfur is an important component of GLS. Therefore, the level of S fertilization and the availability of this element have an effect on the synthesis of alkenyl GLS [42,43,44]. A significant increase in the content of GLS in oilseed rape has been observed with S fertilization [14,18,20,23,32,33,42,45]. In particular, the increase in GLS content was more pronounced under conditions of S deficiency and with a higher dose of S fertilization [23]. On the other hand, a smaller increase in GLS with S fertilization was observed under conditions of good S supply [42,46,47].

The utility value of seeds and rapeseed oil is also significantly influenced by environmental factors (availability of water and nutrients, and temperature) [11,48,49]. Researchers point out that moisture conditions [50] and temperature [51,52] during seed development and maturation determine their fat and protein content. In addition to influencing fat and protein content, moisture and thermal conditions during oil deposition and seed maturation also affect the fatty acid profile [32,36,53,54].

Variability in the GLS content of winter oilseed rape seeds can also be attributed to environmental factors such as temperature, water stress [8,55,56,57] and soil type [48,58,59]. GLS content is affected by both water deficiency and excess water supply [56]. Drought reduces S uptake and thus affects GLS synthesis, whereas excess water accelerates plant growth and reduces GLS content by diluting or leaching S from the soil [8,56].

The aim of this study was to determine the effect of S fertilization under different N doses on the content of fat, protein and GLS in seeds and the proportion of fatty acids in the oil obtained from new forms of rapeseed characterized by an altered fatty acid profile.

2. Materials and Methods

2.1. Plant Material

The plant material used in this study included the canola-type winter oilseed rape cultivar Monolit with a typical C18 fatty acid composition: oleic acid (C18:1, 63.0%), linoleic acid (C18:2, 20.0%) and linolenic acid (C18:3, 9.0%); the cultivar Polka (HO) with high oleic acid content (C18:1, 78.0%) and low linoleic acid content (C18:2, 7.6%) and linolenic acid content (C18:3, 7.8%); and the breeding genotype PN440 (HOLL) with high oleic acid content (C18:1, 75.0), linoleic acid content (C18:2, 13.0%) and low linolenic acid content (C18:3, 3.0%). The modified winter oilseed rape genotypes (HO and HOLL) with different fatty acid profiles were obtained by chemically induced mutagenesis [6] at the Plant Breeding and Acclimatization Institute—National Research Institute, in Poznań, Poland.

2.2. Field Experiment

The field experiment was conducted in Łagiewniki (51°46′ N, 17°14′ E, 104 m a.s.l.) in western Poland in three growing seasons, 2015/16, 2016/17 and 2017/18. The study was set up in a system of randomized sub-blocks, in four replications, in which were tested, in the following order: (N) three spring doses of N (100, 160 and 220 kg N ha⁻¹) in the form of ammonium nitrate; (S) four spring doses of S (0, 30, 60 and 90 kg S ha⁻¹) in the form of ammonium sulphate; and (C) three winter oilseed rape genotypes characterized by different C18 unsaturated fatty acid contents.

The trials were carried out each year on real brown soils of heavy loamy sand, light or medium loam of quality class IIIa and good wheat complex. The pH of the soil ranged from 6.3 to 7.2, measured with 1 M KCl. The soil had high concentrations of available phosphorus ranging from 214 to 335 mg, P₂O₅ kg⁻¹ soil and potassium from 199 to 296 mg, K₂O kg⁻¹ soil and also magnesium from 79 to 105 mg, Mg kg⁻¹ soil, but low content of assimilable S. The content of S-SO₄ in the arable layer ranged from 98 to 110 mg, S-SO₄ kg⁻¹ soil. The preceding crops were cereals, namely spring barley (2015/2016), winter wheat (2016/2017) and winter rye (2017/2018). Winter oilseed rape (70 seeds/m² at 30 cm spacing) was sown annually on agrotechnical dates (27, 29 and 28 August) on plots of 13.2 (to harvest 9.6) m². After sowing, weeds were controlled each year with a mixture of metazachlor + quinomerac and dimethachlor + napropamide + clomazone. In the autumn of the first year, volunteer cereals and monocotyledonous weeds were controlled with chisalophop-P-ethyl. For pest control, lambda-cyhalothrin and acetamiprid were applied twice in the first and second year, and acetamiprid twice in the last year. In the autumn of the first and second year, tebuconazole was used to protect the crops from disease. Fungicides containing active ingredients from the group of anilides + strobilurins or benzimidazoles were used in the autumn.

Before sowing, the plots were fertilized with 30 kg N ha⁻¹, 53 kg P ha⁻¹ and 105 kg K ha⁻¹. In spring, S was applied as ammonium sulfate (21% N and 24% S) and N as ammonium nitrate (34% N) (−S) or ammonium nitrate and ammonium sulfate (+S). Both compounds were applied at the beginning of the growing season, in phase (BBCH-30). Higher doses of N (160 and 220 kg N ha⁻¹) and S (90 kg S ha⁻¹) were divided into two parts (2/3 and 1/3) and the second part was applied at the beginning of budding (BBCH-51).

Winter oilseed rape was harvested at physiological maturity with a small-plot harvester on the following dates: 20 July 2016, 21 July 2017 and 9 July 2018. The protein and fat content and the fatty acid composition, as well as the quantity and quality of GLS, were analysed in the harvested seeds.

2.3. Biochemical Analysis

2.3.1. Determination of Oil and Protein

Oil content was determined by means of nuclear magnetic resonance (NMR) (broadband NMR analyser; Newport Instruments Ltd. Oxford. UK)) [60]. The protein content was estimated by the Kjeldahl method. The calculated amount of nitrogen was multiplied by the conversion factor of 6.25.

2.3.2. Determination of Fatty Acids

Fatty acid composition was determined by gas chromatography using an Agilent Technologies 6890N Network GC System(Santa Clara, CA, USA). Samples of 0.1 to 0.3 g of dried seeds were ground in the grinder and transferred to scintillation vials. Each vial was filled with 2 mL of hexane. The vials were then placed in a shaker with the shaking speed adjusted to allow grinding of one sample. After 15 min of grinding, followed by centrifugation, the hexane solution was transferred to a glass vial, where it was stratified to produce fatty acid methyl esters (FAMEs). The separation of the esters was carried out using a DB-23 capillary column 30 m in length, with hydrogen as the carrier gas, a column temperature of 200 °C and a detector temperature of 220 °C. The separation time was approximately 10 min. The course of the chromatographic separation was recorded and the percentage of each fatty acid was calculated using ChemStation software (Santa Clara, CA, USA).

2.3.3. Determination of Glucosinolates

Glucosinolate content and composition were determined by gas chromatography. Glucosinolates were extracted from seeds using methanol with barium acetate. Silyl derivatives of desulfoglucosinolates were then obtained and the total glucosinolate content (expressed as μM g⁻¹ seeds) was analysed. In this method, the European standard CRM-366, with a total glucosinolate content of 12.1 μM g⁻¹ seeds and a tolerance of 0.8 μM g⁻¹ seeds, was used to calibrate the chromatograph. This standard was developed by the Community Bureau of Reference (BCR) as the mean of interlaboratory comparisons between eighteen laboratories.

2.4. Statistical Analysis

Data were statistically analysed using the STATISTICA package(v. 7.1). The significance of differences between means was assessed using Tukey’s HSD test (p < 0.05).

3. Results

3.1. Weather Conditions

The temperature and humidity conditions during the winter rapeseed vegetation period differed significantly from the multi-year averages in all the years studied. The average temperature during the winter dormancy period (December–March) was significantly higher (by 2.3 and 0.9 °C in the first and second years, respectively) or slightly higher (by 0.2 °C in the last year) than the long-term averages. In the first year of the study, the sum of precipitation during this period was higher than average (by about 27 mm, 20%), while in the remaining years it was more than a quarter lower (Figure 1). The earliest resumption of vegetation was observed in the second year of the study (2 March), i.e., after 111 days of winter dormancy. Vegetation started later in the first (30 March) and third (7 April) years, with winter dormancy lasting 125 and 136 days, respectively.

In the study years, the temperature during the spring–summer vegetation period, from resprouting to seed maturity, was higher (by 0.3–4.9 °C) than the monthly mean daily air temperature, with an unfavourable rainfall distribution. The mean daily air temperature during this vegetation period (April–June) was 1.2 °C higher than the long-term mean in the first year, and up to 3.3 °C higher in the last year of the experiment, while in the second year it was only slightly higher (by 0.3 °C) than the long-term mean (Figure 1). Total rainfall in April of the first and second years was more than 17 mm (55%) and 8 mm (25%) higher than the long-term average, respectively, while it was more than 30% lower (18 and 22 mm) during the flowering phase (in May). During the silique-development and seed-maturation phases, i.e., in June and the first ten days of July, rainfall was close to the norm only in the first year, while in the other two years it was more than 30% (22 and 19 mm) lower. In the last year of the study, April temperatures were well above average (up to 4.9 °C) and precipitation was 60% (19 mm) below the long-term mean. Similarly, in the remaining months (May, June, and July) of the spring- and summer-vegetation period, temperatures were much higher than average (by 3.0, 2.0 and 2.0 °C, respectively) and precipitation much lower than normal (especially in May and June, by 38 mm, 67%, and 19 mm, 29%, respectively). In only the first year of the study. rainfall was recorded from April to July at a level of about 240 mm, which was sufficient to meet the water requirements of rape (225 mm). In the other two cycles of the study period, rainfall was lower by 33 mm and 62 mm. respectively.

3.2. Total-Protein and Crude-Fat Content

The ANOVA F-test statistics for three factors (N—nitrogen rates; S—sulfur rates; C—cultivar) and their interactions over years (Y) are presented in

Table 1. ANOVA F-test statistics.

Parameter	Y	N	S	C	Y × N	Y × S	N × S	Y × C	N × C	S × C	Y × N × S	N × S × C	Y × N × C	Y × S × C	Y × N × S × C
Content of seeds [g kg−1 DM]
crude fat	497.14 **	15.99 **	2.98 *	284.29 **	0.22 ns	1.26 ns	2.42 *	20.95 **	1.20 ns	0.48 ns	0.89 ns	0.93 ns	0.55 ns	0.52 ns	0.87 ns
total protein	159.81 **	27.69 **	5.40 **	240.67 **	1.71 ns	1.26 ns	3.35 **	4.37 **	1.55 ns	0.47 ns	0.74 ns	1.38 ns	1.92 ns	0.37 ns	0.87 ns
Fatty acids [%]:
C16	38.91 **	0.30 ns	0.67 ns	4626.51 **	0.05 ns	2.41 *	1.03 ns	34.16 **	1.62 ns	0.97 ns	1.04 ns	0.82 ns	0.60 ns	1.46 ns	1.52 ns
C18	328.47 **	1.74 ns	3.96 *	67.35 **	1.96 ns	1.45 ns	0.90 ns	41.2 **	0.71 ns	1.61 ns	0.70 ns	0.57 ns	1.71 ns	0.76 ns	0.71 ns
C18:1	121.15 **	0.34 ns	1.06 ns	8071.52 **	4.45 *	1.36 ns	0.57 ns	71.00 **	0.48 ns	1.96 ns	1.03 ns	1.34 ns	0.61 ns	1.29 ns	0.97 ns
C18:2	122.97 **	0.85 ns	1.45 ns	8098.36 **	1.49 ns	1.04 ns	0.85 ns	67.05 **	0.75 ns	1.13 ns	0.84 ns	1.39 ns	0.40 ns	1.45 ns	1.06 ns
C18:3	426.96 **	2.29 ns	0.95 ns	1630.55 **	2.74 ns	0.95 ns	0.35 ns	25.36 **	0.30 ns	0.92 ns	1.39 ns	0.98 ns	1.08 ns	0.44 ns	0.71 ns
C20:1	18.82 **	14.86 **	0.59 ns	953.84 **	0.40 ns	1.07 ns	0.12 ns	3.88 **	2.30 ns	0.57 ns	1.15 ns	0.55 ns	0.51 ns	0.64 ns	0.92 ns
C22:1	2.20 ns	3.39 ns	0.16 ns	1.89 ns	1.57 ns	0.99 ns	0.69 ns	0.57 ns	0.84 ns	1.26 ns	0.48 ns	1.43 ns	0.60 ns	1.04 ns	0.86 ns
SFA	71.35 **	0.99 ns	2.98 *	3037.88 **	0.44 ns	0.75 ns	1.14 ns	47.25 **	0.55 ns	0.97 ns	1.06 ns	0.69 ns	1.02 ns	1.26 ns	1.05 ns
UFA	60.66 **	0.59 ns	1.67 ns	663.55 **	0.29 ns	1.06 ns	0.84 ns	32.25 **	1.28 ns	1.04 ns	0.80 ns	0.80 ns	0.61 ns	0.85 ns	1.02 ns
MUFA	120.91 **	0.39 ns	1.19 ns	8586.24 **	3.87 *	1.37 ns	0.56 ns	72.18 **	0.60 ns	1.77 ns	1.13 ns	1.25 ns	0.53 ns	1.17 ns	1.39 ns
PUFA	188.16 **	0.27 ns	1.03 ns	7587.9 **	3.68 *	1.49 ns	0.43 ns	80.02 **	0.45 ns	1.67 ns	1.09 ns	1.23 ns	0.68 ns	1.23 ns	1.49 ns
Glucosinolate content [μM g−1 seeds]
glnap	68.29 **	7.03 **	20.26 **	157.23 **	0.27 ns	1.44 ns	1.00 ns	51.73 **	0.99 ns	2.61 *	0.72 ns	0.76 ns	0.84 ns	1.63 ns	0.97 ns
glbra	38.46 **	9.12 **	24.83 **	906.02 **	1.19 ns	1.08 ns	0.85 ns	135.09 **	2.48 *	1.67 ns	0.42 ns	0.80 ns	0.61 ns	1.22 ns	1.07 ns
progo	4.54 ns	16.88 **	16.14 **	36.47 **	0.65 ns	0.58 ns	1.36 ns	44.46 **	2.01 ns	2.34 *	0.55 ns	0.76 ns	1.07 ns	1.63 ns	0.93 ns
napol	2.27 ns	2.38 ns	14.74 **	320.82 **	0.90 ns	1.10 ns	0.90 ns	23.48 **	3.49 **	7.01 **	0.52 ns	0.76 ns	0.34 ns	2.06 *	1.56 ns
ind	1.40 ns	1.51 ns	1.56 ns	21.01 **	1.20 ns	0.49 ns	0.78 ns	8.53 **	1.81 ns	0.84 ns	0.98 ns	1.14 ns	1.50 ns	0.49 ns	0.64 ns
4-OH	5.66 *	3.21 *	0.67 ns	36.67 **	0.71 ns	0.83 ns	0.65 ns	5.51 **	1.36 ns	1.28 ns	0.66 ns	0.31 ns	0.36 ns	0.51 ns	1.14 ns
alkenyl	15.2 **	13.27 **	20.40 **	29.67 **	0.45 ns	0.60 ns	1.20 ns	61.57 **	0.98 ns	2.69 *	0.63 ns	0.64 ns	0.94 ns	1.72 ns	0.96 ns
indole	5.31 *	3.61 *	0.56 ns	35.61 **	0.78 ns	1.05 ns	0.62 ns	6.14 **	1.36 ns	1.17 ns	0.65 ns	0.32 ns	0.42 ns	0.52 ns	1.09 ns
total	1.35 ns	13.28 **	11.89 **	52.04 **	0.81 ns	0.68 ns	1.17 ns	24.71 **	0.08 ns	2.66 *	0.82 ns	0.64 ns	0.61 ns	1.33 ns	1.08 ns

Y—growing season; N—spring nitrogen rates (kg ha⁻¹); S—spring sulfur rates (kg ha⁻¹); C—cultivar. C16—palmitic acid; C18—stearic acid; C18:1—oleic acid; C18:2—linoleic acid; C18:3—linolenic acid; C20:1—eicosenoic acid; C22:1—erucic acid; SFA—saturated fatty acids (C16 + C18); UFA—unsaturated fatty acids (C18:1 + C18:2 + C18:3); MUFA—monounsaturated fatty acids (C18:1 + C20:1 + C22:1); PUFA—polyunsaturated fatty acids (C18:2 + C18:3). glnap—gluconapin; glbra—glucobrassicanapin; progo—progoitrin; napol—napoleiferin; ind—indol; 4-OH—4-hydroksyglucobrassicin; * significant p < 0.05. ** significant p < 0.01; ns—not significant.

. The results for the individual experiments, as well as the 3-year results of the research, showed that the analysed fertilization factors significantly influenced the fat and protein content of the seeds of the tested winter oilseed rape varieties. The response of the investigated varieties to the fertilization factors was similar.

No significant interaction was observed between the variety and the doses of N and S fertilization. The results, averaged over three years, showed that an increase in N fertilization doses from (100 kg ha⁻¹) to (160 and 220 kg ha⁻¹) reduced crude fat content (by 6.5–10.5 g kg⁻¹ dry matter (DM)) and increased total protein content (by 3.2–10.1 g kg⁻¹ DM) in all types of investigated winter oilseed rape. However, significant changes in fat and protein content were only observed up to a nitrogen dose of 160 kg N ha⁻¹. The dose of S fertilization applied (30 kg ha⁻¹) resulted in a significant increase in fat content (by 3.3 g kg⁻¹ DM) and a decrease in protein content (by 3.0 g kg⁻¹ DM) in the seeds of all winter oilseed rape varieties tested, compared with the control (0 kg S dose) (

Table 2. Significance of differences between the mean values of the main effects: year (Y) and three studied factors (N, S, C) in an analysis of the crude fat and total protein in the seeds and fatty acid content in the oil investigated, of winter oilseed rape genotypes.

	Content
Factor/Level	g kg−1 DM		Fatty Acids [%]
	Crude Fat	Total Protein	C16	C18	C18:1	C18:2	C18:3	C20:1	C22:1	SFA	UFA	MUFA	PUFA
2015/2016	469.0 a†	208.2 c	4.18 a	1.65 c	71.6 c	13.7 a	7.65 a	1.20 b	0.01	5.83 c	92.9 a	72.8 c	21.4 a
2016/2017	461.5 b	223.4 b	3.91 b	2.12 b	74.8 a	11.2 c	6.66 c	1.27 a	0.00	6.03 b	92.7 b	76.1 a	17.9 c
2017/2018	448.7 c	227.9 a	3.93 b	2.29 a	72.9 b	12.6 b	6.96 b	1.31 a	0.01	6.22 a	92.5 c	74.2 b	19.6 b
N100	465.7 a	214.2 b	4.02	2.03	73.1	12.5	7.15	1.23 b	0.00	6.05	92.7	74.3	19.6
N160	459.2 b	221.1 a	4.00	2.03	73.1	12.5	7.06	1.26 a	0.01	6.03	92.7	74.4	19.6
N220	455.2 b	224.3 a	4,00	2.01	73.1	12.6	7.06	1.28 a	0.02	6.00	92.7	74.4	19.6
S0	458.4 b	221.6 a	4.02	2.04 a	73.0	12.6	7.05	1.26	0.01	6.06 a	92.7	74.3	19.7
S30	461.7 a	218.6 b	4.01	2.02 ab	73.2	12.4	7.09	1.27	0.01	6.02 ab	92.7	74.4	19.5
S60	459.5 ab	219.4 b	4.00	2.00 b	73.1	12.5	7.13	1.25	0.01	6.00 b	92.7	74.3	19.7
S90	460.5 ab	219.9 ab	4,00	2.02 ab	73.2	12.4	7.08	1.25	0.01	6.02 ab	92.7	74.4	19.5
Monolit	470.9 a	212.3 b	4.67 a	2.07 a	66.3 c	17.7 a	8.21 a	1.02 c	0.01	6.74 a	92.2 c	67.3 c	25.9 a
Polka	450.0 c	222.9 a	3.42 c	1.97 c	77.3 a	8.25 c	7.55 b	1.51 a	0.00	5.39 c	93.1 a	78.8 a	15.8 c
PN440	459.2 b	224.3 a	3.93 b	2.02 b	75.7 b	11.6 b	5.51 c	1.24 a	0.02	5.95 b	92.8 b	76.9 b	17.1 b

Y—growing season; N—spring nitrogen rates (kg ha⁻¹); S—spring sulfur rates (kg ha⁻¹); C—cultivar. DM, dry matter; C16—palmitic acid; C18—stearic acid; C18:1—oleic acid; C18:2—linoleic acid; C18:3—linolenic acid; C20:1—eicosenoic acid; C22:1—erucic acid; SFA—saturated fatty acids (C16 + C18); UFA—unsaturated fatty acids (C18:1 + C18:2 + C18:3); MUFA—monounsaturated fatty acids (C18:1 + C20:1 + C22:1); PUFA—polyunsaturated fatty acids (C18:2 + C18:3). ^† Means with the same letter are not significantly different at p ≤ 0.05, according to Tukey’s HSD test.

). According to the interaction between N and S (Figure 2), such an effect was obtained with a low or moderate dose of N fertilization (100 and 160 kg N ha⁻¹). At the maximum dose of N (220 kg ha⁻¹), the application of S at higher doses (60 and 90 kg ha⁻¹) caused an increase in protein content, but it was not significant. Under these conditions, a decrease in the fat content in the seeds of winter oilseed rape was also observed.

Regardless of fertilization factors, weather conditions—temperature and precipitation (research years)—strongly influenced crude-fat and total-protein content. The highest-fat and lowest-protein content were observed in seeds collected in the first year of the research (2016), i.e., when the water supply to the plants was sufficient during the period of oil accumulation and seed maturation. The lowest-fat and highest-protein contents were observed in seeds collected in the last year (2018), when there was an extremely high water deficit during the spring–summer vegetation period (Table 2).

Crude-fat and total-protein content differed significantly among the three winter oilseed rape genotypes studied. In all the years, the seeds of the genotypes with an changed fatty acid profile Polka (HO) and PN440 (HOLL) accumulated significantly more protein (222.9 and 224.3 g kg⁻¹ DM, respectively) than those of the cultivar Monolit, with a typical fatty acid composition (212.3 g kg⁻¹ DM) which was characterized by the highest fat content (470.9 g kg⁻¹ DM) (Table 2). During the spring and summer growing season (2016, when water was abundant, the fat and protein contents of Polka (HO) and PN440 (HOLL) winter oilseed rape were not significantly different, whereas during water-deficit years (low rainfall, 2017 and 2018) seeds of the PN440 (HOLL) genotype accumulated significantly more fat and protein in comparison to the Polka cultivar (

Table 3. Significance of differences between mean values of interactions (Y × C, Y × N and Y × S) in evaluation of crude-fat and total-protein content in the seeds and fatty acid content in the oil of investigated winter oilseed rape genotypes.

Growing Season	Factor/Level	Content
		g kg−1 DM		Fatty Acids [%]
		Crude Fat	Total Protein	C16	C18	C18:1	C18:2	C18:3	C20:1	C22:1	SFA	UFA	MUFA	PUFA
2015/2016	Monolit	476.8 a†	201.7 f	4.92 a	1.76 e	65.1 g	18.4 a	8.74 a	0.99 f	0.01	6.68 b	92.3 de	66.1 h	27.2 a
	Polka	464.9 cd	211.7 e	3.60 g	1.62 f	75.9 c	9.56 e	7.85 b	1.46 b	0.00	5.22 g	93.3 a	77.4 d	17.4 d
	PN440	468.0 bc	211.4 e	4.03 d	1.56 g	73.8 e	13.1 c	6.35 e	1.16 d	0.03	5.59 e	93.2 ab	74.9 f	19.5 c
2016/2017	Monolit	471.4 b	216.7 d	4.61 b	2.14 c	67.1 f	17.2 b	8.00 b	1.0 f	0.00	6.75 ab	92.2 ef	68.1 g	25.2 b
	Polka	451.7 e	225.5 c	3.30 i	2.04 d	78.8 a	7.11 g	7.22 d	1.52 a	0.00	5.34 f	93.1 b	80.3 a	14.3 f
	PN440	461.5 d	228.0 b	3.81 f	2.18 c	78.6 a	9.38 e	4.76 g	1.28 c	0.00	6.00 d	92.8 c	79.9 b	14.1 f
2017/2018	Monolit	464.6 cd	218.6 d	4.48 c	2.31 a	66.8 f	17.5 b	7.89 b	1.07 e	0.02	6.79 a	92.1 f	67.8 g	25.4 b
	Polka	433.4 g	231.7 a	3.36 h	2.25 b	77.2 b	8.08 f	7.58 c	1.55 a	0.00	5.61 e	92.8 c	78.7 c	15.7 e
	PN440	448.1 f	233.5 a	3.95 e	2.31 a	74.7 d	12.3 d	5.42 f	1.29 c	0.02	6.26 c	92.4 d	76.0 e	17.7 d
2015/2016	N100	474.5	204.7	4.19	1.64	71.7 d	13.60	7.67	1.17	0.00	5.83	93.0	72.9 d	21.3 a
	N160	470.0	208.2	4.19	1.64	71.5 d	13.74	7.66	1.21	0.02	5.83	92.9	72.8 d	21.4 a
	N220	465.3	211.8	4.17	1.66	71.5 d	13.79	7.61	1.23	0.03	5.83	92.9	72.8 d	21.4 a
2016/2017	N100	468.2	216.0	3.92	2.14	74.7 a	11.20	6.80	1.24	0.00	6.06	92.7	75.9 b	18.0 d
	N160	459.6	227.0	3.90	2.12	74.8 a	11.29	6.66	1.27	0.00	6.01	92.7	76.0 ab	17.9 de
	N220	456.8	227.2	3.91	2.10	75.0 a	11.18	6.51	1.29	0.00	6.01	92.7	76.3 a	17.7 e
2017/2018	N100	454.6	221.8	3.95	2.30	72.9 bc	12.63	6.98	1.29	0.00	6.25	92.5	74.2 c	19.6 bc
	N160	448.0	228.1	3.93	2.32	73.1 b	12.50	6.88	1.30	0.00	6.25	92.5	74.4 c	19.4 c
	N220	443.5	234.0	3.92	2.26	72.7 c	12.72	7.04	1.33	0.04	6.18	92.4	74.1 c	19.8 b
2015/2016	S0	469.1	208.8	4.22 a	1.65	71.5	13.8	7.61	1.18	0.00	5.88	92.9	72.7	21.4
	S30	473.6	206.6	4.19 ab	1.66	71.7	13.6	7.60	1.22	0.02	5.84	92.9	73.0	21.2
	S60	468.9	208.8	4.16 b	1.64	71.4	13.8	7.72	1.21	0.03	5.80	92.9	72.7	21.5
	S90	468.1	208.9	4.16 b	1.64	71.7	13.7	7.66	1.20	0.01	5.81	93.0	72.9	21.3
2016/2017	S0	459.9	225.2	3.92 cd	2.15	74.6	11.4	6.69	1.27	0.00	6.07	92.7	75.8	18.1
	S30	462.1	222.4	3.92 cd	2.10	74.8	11.3	6.66	1.27	0.00	6.02	92.7	76.1	17.9
	S60	461.2	223.3	3.91 d	2.10	74.8	11.2	6.71	1.25	0.00	6.01	92.7	76.1	17.9
	S90	462.9	222.7	3.89 c	2.13	75.1	11.0	6.59	1.26	0.00	6.02	92.7	76.4	17.6
2017/2018	S0	446.2	230.9	3.92 cd	2.32	72.9	12.6	6.87	1.32	0.03	6.23	92.4	74.3	19.5
	S30	449.5	226.7	3.91 d	2.30	72.9	12.5	7.02	1.31	0.00	6.21	92.5	74.2	19.6
	S60	448.4	226.1	3.94 cd	2.27	72.9	12.7	6.98	1.29	0.00	6.20	92.5	74.2	19.6
	S90	450.6	228.0	3.97 c	2.28	72.8	12.7	6.99	1.30	0.03	6.25	92.4	74.1	19.7

C16—palmitic acid; C18—stearic acid; C18:1—oleic acid; C18:2—linoleic acid; C18:3—linolenic acid; C20:1—eicosenoic acid; C22:1—erucic acid; SFA—saturated fatty acids (C16 + C18); UFA—unsaturated fatty acids (C18:1 + C18:2 + C18:3); MUFA—monounsaturated fatty acids (C18:1 + C20:1 + C22:1); PUFA—polyunsaturated fatty acids (C18:2 + C18:3). ^† Means with the same letter are not significantly different at p ≤ 0.05, according to Tukey’s HSD test.

).

3.3. Composition of Fatty Acids in Oil

The applied N and S fertilization doses had no significant effect on the content of the main unsaturated fatty acids (UFAs) [(oleic (C18:1) + linoleic (C18:2) + linolenic (C18:3)] in winter rape oil (Table 2). The fatty acid content of the oil depended mainly on the winter oilseed rape genotypes.

The content of fatty acids in the oil of the winter oilseed rape genotypes studied was found to be strongly influenced by the weather conditions—temperature and precipitation—during the stages of flowering, silique development and seed maturation (Table 2). Under conditions of good water supply and moderate temperature during the period of oil accumulation and seed maturation (2016), the oil of the studied genotypes contained significantly higher palmitic acid (C16), by 0.27% and 0.25%, compared to 2017 and 2018 respectively, linoleic acid (C18:2), by 2.5% and 1.1%, and linolenic acid (C18:3) by 0.99% and 0.69%, as well as a higher sum of 18-carbon unsaturated fatty acids (UFAs) by 0.2% and 0.4%, and polyunsaturated fatty acids (PUFAs), by 3.5% and 1.8%. The lack of precipitation and the higher temperatures observed in the second (2017,) and especially in the third (2018), year of the study caused a significant increase in the content of stearic acid (C18), by 0.47% and 0.64%, respectively, oleic acid (C18:1), by 3.2% and 1.3% respectively, and eicosenoic acid (C20:1) by 0.07% and 0.11%, as well as an increase in the sum of saturated fatty acids (SFAs), by 0.2% and 0.39%, and the sum of monounsaturated fatty acids (MUFAs), by 3.3% and 1.4% (Table 3).

The winter oilseed rape genotypes studied differed in their responses to environmental parameters. Greater changes in the oil, with significantly greater (up to several times) decreases or increases in the content of individual fatty acids, were observed in the genotypes with altered fatty acid profiles (Table 3). In particular, in genotype PN440 (HOLL), the sum of 18-carbon unsaturated fatty acids (UFAs) decreased by 0.4–0.8%, especially linoleic acid (C18:2), by 3.72–0.8%, linolenic acid (C18:3), by 1.59–0.93%, and the content of oleic acid (C18:1), by 4.8–0.9%. The content of saturated fatty acids (SFAs) increased, especially stearic acid (C18), by 0.62–0.75% under drought conditions (2017 and 2018). These changes were significantly higher than those observed in the Monolit cultivar, where unsaturated fatty acids (UFAs) decreased by 0.1–0.2%, linoleic acid (C18:2) by 1.2–0.91%, and linolenic acid (C18:3) by 0.74–0.75%, while oleic acid (C18:1) increased by 2–1.7% and stearic acid (C18) by 0.38–0.55%, compared to 2016.

3.4. Glucosinolate (GLS) Content in Seeds

Data analysis showed that different N and S doses significantly influenced the content of GLS in winter oilseed rape seeds (Table 1). Increasing the N dose from 100 to 160 kg N ha⁻¹ caused a significant increase in the content of alkenyl GLS compounds, namely gluconapin (glnap) and progoitrin (progo), as well as a significant increase in the sum of alkenyl and total GLS. The application of the maximum N dose (220 kg ha⁻¹) did not cause a significant increase in these GLS contents; on the other hand, it significantly increased the contents of glucobrassicanapin (glbra) and indole GLS (4-hydroxyglucobrassicin (4-OH), total indole) (

Table 4. Effect of experimental factors on GLS content in seeds of investigated winter oilseed rape genotypes.

Factor/Level	Glucosinolate Content [μM g−1 Seeds]
	glnap	glbra	progo	naplo	indol	4-OH	Alkenyl GLS		Indole GLS		Total GLS
								rel		rel		rel
2015/2016	1.84 c†	0.63 b	4.38	0.19	0.11	5.59 a	7.04 b	100	5.70 a	100	12.7	100
2016/2017	2.07 b	0.85 a	4.76	0.16	0.10	4.98 b	7.84 a	112	5.08 b	89	12.9	102
2017/2018	2.53 a	0.80 a	4.73	0.12	0.11	4.89 b	8.18 a	116	5.00 b	88	13.2	104
N100	2.06 b	0.72 b	4.34 b	0.16	0.10	5.03 b	7.28 b	100	5.13 b	100	12.4 b	100
N160	2.18 a	0.76 ab	4.70 a	0.15	0.10	5.14 ab	7.79 a	107	5.24 ab	102	13.0 a	105
N220	2.21 a	0.79 a	4.82 a	0.17	0.11	5.30 a	7.99 a	110	5.41 a	106	13.4 a	108
S0	2.04 c	0.68 c	4.36 c	0.13 b	0.10	5.14	7.21 c	100	5.24	100	12.4 b	100
S30	2.12 bc	0.75 b	4.59 b	0.16 a	0.11	5.22	7.62 b	106	5.33	102	12.9 a	104
S60	2.18 ab	0.79 ab	4.69 ab	0.17 a	0.11	5.10	7.83 ab	109	5.21	100	13.0 a	105
S90	2.25 a	0.81 a	4.84 a	0.17 a	0.11	5.15	8.07 a	112	5.26	101	13.3 a	107
Monolit	1.91 c	0.45 c	4.89 a	0.22 a	0.09 b	5.07 b	7.47 b	100	5.16 b	100	12.6 b	100
Polka	2.38 a	1.02 a	4.53 b	0.14 b	0.11 a	5.49 a	8.09 a	108	5.60 a	108	13.7 a	109
PN440	2.15 b	0.80 b	4.44 b	0.11 c	0.12 a	4.90 c	7.50 b	100	5.02 b	97	12.5 b	99

Glnap—gluconapin; glbra—glucobrassicanapin; progo—progoitrin; naplo—napoleiferin; 4-OH—4-hydroksyglucobrassicin. ^† Means with the same letter are not significantly different at p ≤ 0.05, according to Tukey’s HSD test.

). A significant increase in glbra content was observed only in the cultivar Polka and line PN440 (Table 4). Similar to N, an increase in S doses resulted in a significant increase in the content of alkenyl GLS (glnap, glbra, progo and napoleiferin (naplo)) and total GLS, but no significant increase in the content of indole GLS. Both N and S fertilization resulted in a greater increase in total alkenyl GLS (10% and 12%, respectively) than in total indole GLS (6% and 2%, respectively). There was no significant interaction between N and S.

The significant interaction between winter oilseed rape genotypes and S doses was evident for the content of alkenyl GLS (glnap, progo and naplo) and the sum of alkenyl and total GLS (Table 1). In the cultivars Monolit and Polka, the content of the two most harmful alkenyl GLSs, glnap and progo, increased significantly with increasing S dose up to the maximum value (90 kg S ha⁻¹), while in the line PN440 an increase in GLS content was observed up to a dose of 60 kg S ha⁻¹ (

Table 5. Significance of differences between mean values of interactions (S × C, N × C and Y × C) in evaluation of glucosinolate (GLS) content [μM g⁻¹ seeds] in seeds of investigated winter oilseed rape genotypes.

Factor/Level		Glucosinolate Content [μM g−1 seeds]
		glnap	glbra	progo	naplo	indol	4-OH	Alkenyl GLS		Indole GLS		Total GLS
									rel		rel		rel
Monolit	S0	1.83 h†	0.38	4.61 c	0.17 b	0.09	5.02	6.99 e	100	5.11	100	12.1 f	100
	S30	1.88 h	0.46	4.92 ab	0.24 a	0.10	5.23	7.50 cd	107	5.33	104	12.8 cde	106
	S60	1.91 gh	0.45	4.90 b	0.24 a	0.10	4.93	7.50 cd	107	5.02	98	12.5 ef	103
	S90	2.02 fg	0.48	5.15 a	0.24 a	0.09	5.11	7.89 bc	113	5.20	102	13.1 cd	108
Polka	S0	2.22 cd	0.93	4.22 d	0.12 def	0.11	5.47	7.49 cd	100	5.58	100	13.1 cde	100
	S30	2.31 bc	1.02	4.41 cd	0.15 bc	0.11	5.42	7.89 bc	105	5.51	99	13.4 bc	102
	S60	2.43 b	1.05	4.60 c	0.14 cd	0.11	5.55	8.22 b	110	5.66	101	13.9 ab	106
	S90	2.57 a	1.10	4.91 ab	0.17 b	0.11	5.54	8.75 a	117	5.65	101	14.4 a	110
PN440	S0	2.06 ef	0.76	4.24 d	0.09 g	0.11	4.92	7.15 de	100	5.03	100	12.2 f	100
	S30	2.15 def	0.77	4.45 cd	0.11 efg	0.11	5.04	7.48 cd	105	5.15	102	12.6 def	103
	S60	2.20 cd	0.87	4.58 c	0.13 cde	0.12	4.83	7.78 c	109	4.95	98	12.7 def	101
	S90	2.18 cde	0.82	4.48 cd	0.10 fg	0.12	4.81	7.58 c	106	4.93	98	12.5 ef	102
Monolit	N100	1.78	0.44 e	4.49	0.23 a	0.09	5.03	6.94	100	5.12	100	12.1	100
	N160	1.94	0.45 e	5.03	0.21 a	0.09	5.04	7.63	110	5.13	100	12.8	106
	N220	1.99	0.45 e	5.18	0.22 a	0.10	5.17	7.84	113	5.27	103	13.1	109
Polka	N100	2.33	0.98 b	4.34	0.14 bc	0.11	5.28	7.79	100	5.38	100	13.2	100
	N160	2.42	1.03 ab	4.61	0.13 c	0.11	5.44	8.19	105	5.55	103	13.7	104
	N220	2.41	1.07 a	4.65	0.16 b	0.11	5.75	8.29	106	5.88	109	14.2	108
PN440	N100	2.06	0.75 d	4.19	0.10 d	0.12	4.77	7.10	100	4.89	100	12.0	100
	N160	2.17	0.79 cd	4.48	0.10 d	0.11	4.95	7.54	106	5.06	103	12.6	105
	N220	2.22	0.86 c	4.65	0.12 cd	0.12	4.97	7.85	111	5.09	104	12.9	108
2015/2016	Monolit	1.73 g	0.49 c	4.73 cd	0.28 a	0.09 d	5.29 bc	7.23 d	100	5.38 c	100	12.6 c	100
	Polka	2.20 cd	0.98 b	4.73 cd	0.19 c	0.10 cd	5.95 a	8.10 b	112	6.05 a	113	14.2 a	113
	PN440	1.58 h	0.43 cd	3.69 f	0.10 ef	0.13 a	5.55 b	5.80 e	80	5.68 b	106	11.5 d	91
2016/2017	Monolit	1.85 f	0.44 cd	5.16 a	0.23 b	0.10 cd	5.12 cd	7.68 c	100	5.22 cd	100	12.9 c	100
	Polka	2.31 c	1.12 a	4.50 de	0.14 d	0.10 cd	5.28 bc	8.07 b	105	5.38 c	103	13.4 b	104
	PN440	2.03 e	0.99 b	4.62 cd	0.12 de	0.10 cd	4.58 e	7.78 bc	101	4.67 e	90	12.4 c	96
2017/2018	Monolit	2.15 d	0.41 d	4.79 bc	0.17 c	0.09 d	4.83 de	7.52 cd	100	4.92 de	100	12.4 c	100
	Polka	2.64 b	0.98 b	4.38 e	0.10 ef	0.13 a	5.25 c	8.10 b	108	5.38 c	109	13.5 b	109
	PN440	2.82 a	0.99 b	5.01 ab	0.09 f	0.12 b	4.57 e	8.91 a	119	4.69 e	95	13.6 b	110

Glnap—gluconapin; glbra—glucobrassicanapin; progo—progoitryn; naplo—napoleiferin; 4-OH—4-hydroksyglucobrassicin. ^† Means with the same letter are not significantly different at p ≤ 0.05, according to Tukey’s HSD test.

). In the Monolit cultivar, the increase in the content of naplo and the sum of alkenyl GLS was observed only up to the dose of 30 kg S ha⁻¹, while in the modified genotypes (Polka cultivar and PN440 line) the increase was observed up to 90 and 60 kg S ha⁻¹, respectively. Total GLS also increased with increasing S dose, but the increase was not significant in line PN440. The highest increase in the sum of alkenyl GLS (by 17%) and total GLS (by 10%) was observed in the cultivar Polka, while the lowest increase (by 9% and 3%, respectively) was found in genotype PN440.

The genotypes differed significantly in their GLS content (Table 4). Regardless of the dose of N and S, the highest sum of alkenyl and indole GLS and total GLS was found in the cultivar Polka (8.09, 5.60 and 13.7 µM g⁻¹ seed, respectively). Significantly less GLS was found in the cultivar Monolit (8%, 8% and 8%, respectively) and genotype PN440 (7%, 10% and 9%, respectively), which were not significantly different from each other. The winter oilseed rape genotypes studied were characterized by a similar proportion of alkenyl GLS (59–60%) and a small proportion of less-harmful indole GLS (40–41%). Among the alkenyl GLSs, progo (56–65%) and glnap (26–29%) were dominant, while the predominant indole GLS was 4-OH (97–98%). In the mutant genotypes with altered fatty acid composition (Polka, PN440), the proportion of progo (56.1% and 59.2%) and naplo (1.7% and 1.5%) was slightly lower than in the rapeseed cultivar Monolit (progo 65.5%, naplo 2.9%), while that of glnap (29.5% and 28.7%) and glbra (12.6% and 10.7%) was slightly higher (glnap 25.6% and glbra 6%). The content of glnap, glbra and 4-OH, as well as the sum of alkenyl and indole GLS, differed significantly between years (Y). In the first year of the study (2016), the content of glnap and glbra and the sum of alkenyl GLS were significantly lower, while the content of 4-OH and the sum of indole GLS were significantly higher compared to the remaining years (2017 and 2018). A significant C × Y interaction was also observed for the content of GLS (Table 5). Greater changes in GLS content over the years studied were observed in the genotypes with altered fatty acid composition. The change was particularly pronounced in the case of genotype HOLL (PN440), whose seeds were characterized by a significantly lower content of GLS in 2016 and the highest content of alkenyl GLS (especially progo, glnap and total alkenyl) and total GLS in 2018. A slight or not-significant difference in GLS content (glbra, indole, total) between the years was found in the cultivar Monolit. All winter oilseed rape genotypes tested had significantly higher total indole GLS in the first year of the study.

4. Discussion

4.1. Total-Protein and Crude-Fat Content

All investigated winter oilseed rape genotypes responded similarly to the fertilization factors (N and S). A significant increase in protein content and a decrease in crude fat content were observed in the seeds of the studied genotypes only up to the N dose of 160 kg N ha⁻¹, while the application of a higher dose (220 kg N ha⁻¹) did not cause any significant change in the content of both components. Many studies have shown a similar relationship, i.e., a reduction in fat content accompanied by an increase in protein content in winter oilseed rape [12,13,14,15,23,61,62]. However, the changes in protein and fat content observed in winter oilseed rape under the influence of N fertilization were small, and did not exceed a few percentage points [52]. In the present study, the changes observed in these components as a result of increasing the N dose from 100 to 220 kg ha⁻¹ did not exceed 10 g kg⁻¹ DM (1 percentage point). Ahmed et al. [18] showed that S, in contrast to N, had a positive effect on the fat content of oilseed rape. Similarly, Sattar et al. [27] observed a significant increase in fat content in their studies. Some studies have also shown an increase in protein content with sulfur fertilization [28,44]. Fismes et al. [63] and Egesel et al. [64] showed that improved S supply to rapeseed plants resulted in a decrease in the oil yield of winter oilseed rape. However, the lack of a significant effect of S on fat [49,65] and protein [20] content has also been reported by many authors. The results of the present study indicate that the effect of S fertilization is not unidirectional and that it interacts with N fertilization. The increase in crude fat content and the decrease in protein content in the seeds of the studied genotypes were caused by the application of the minimum dose of S (30 kg S ha⁻¹) with a low and moderate dose of N (100 and 160 kg N ha⁻¹). Conversely, fat and protein contents were influenced by higher S doses (60 and 90 kg S ha⁻¹) under the condition of high N fertilization (220 kg N ha⁻¹). Zhao et al. [22] also showed that S fertilization at high N doses caused an increase in protein content. The interaction between N and S in determining fat and protein content has also been highlighted by many other researchers [20,28,41]. Sulfur affects N metabolism in plants and plays an important role in protein synthesis, due to its presence in some amino acids and enzymes [36]. The activity of acetyl-CoA carboxylase, an enzyme that catalyses the first step of fatty acid synthesis, increases in the presence of S and N [66]. Previous studies [20] have shown the significant effect of S fertilization on the fat and protein content of oilseed rape in field experiments carried out on S-deficient soils. However, in experiments where S was not a limiting factor for plant growth, no clear effect of S fertilization on fat and protein content was observed [32]. Crude-fat and protein contents were also strongly influenced by environmental parameters, particularly the climatic conditions during the years studied. Researchers [36,50,51,61] have shown that increased soil moisture and moderate temperatures during the seed filling and ripening stages increase oil concentration, while lower soil moisture and high temperatures promote protein accumulation. Faraji [9] demonstrated that prolongation of the seed filling stage and lower temperatures during this period can lead to an increase in oil content. These claims were confirmed by the results of the present study, in which the conditions of good soil moisture during the seed filling stage (2016) allowed the seeds to accumulate more fat (by 20 g kg⁻¹ DM; 2 percentage points), while water deficiency (2017 and 2018) resulted in significantly more protein content (by 20 g kg⁻¹ DM; 2 percentage points) in the seeds. The genotypes with an changed fatty acid profile accumulated significantly more protein and less fat than the standard variety Monolit. The mutual negative relationship between fat and protein content makes it difficult to find a genotype characterized by both high fat and protein content. The physiological cause of the negative correlation between protein and fat may be that an increased nitrogen supply increases protein synthesis at the expense of fatty acid synthesis (the content of available carbohydrates is reduced), and thus reduces the fat content of the seeds [67].

4.2. Composition of Fatty Acids in Oil

The applied N and S fertilization doses had no significant effect on the content of the main unsaturated fatty acids. The available scientific reports on the effect of N fertilization on the fatty acid composition of rapeseed oil are inconclusive. Many of them indicate that N fertilization has no significant effect on the fatty acid composition [68,69,70], while Groth [14] observed a significant increase in the content of C18:3 acid and a decrease in the content of oleic acid (C18:1). Similarly, Wielebski [30] showed a significant increase in the content of eicosenoic acid (C20:1), linoleic acid (C18:2) and linolenic acid (C18:3) and a decrease in the content of oleic acid (C18:1) under the influence of N fertilization. Some authors [52] have attributed the heterogeneity of results describing the effect of N fertilization on fatty acid composition to differences in seed maturity at harvest.

The present study showed that the doses of S had only a minimal effect on the quality of oil obtained from the seeds of new genotypes of winter oilseed rape. Previous studies have also not shown a clear effect of S application on the fatty acid composition of oil obtained from both traditional [71] and double-improved varieties [29,30]. Manaf and Hassan [48] reported a small and variable effect of S fertilization on the proportion of fatty acids in winter oilseed rape oil. Kozłowska-Strawska [72] showed that, with an increase in the abundance of S-SO₄ in soils, the content of oleic acid (C18:1) increased significantly, while that of essential unsaturated fatty acids (UFAs) decreased significantly. The results of the study by Groth [14] reported that the effect of S fertilization is not unidirectional and that it interacts with N fertilization. S fertilization (40 kg S ha⁻¹) with medium N doses (130 kg ha⁻¹) caused a significant increase in the content of oleic acid (C18:1), whereas with high N doses (230 kg ha⁻¹) a significant decrease in the content of oleic acid (C18:1) was observed. However, under the influence of S fertilization, an increase in the proportion of linolenic (C18:3) acid was observed in winter oilseed rape oil fertilized with both low (80 kg ha⁻¹) and very high (230 kg ha⁻¹) N doses. Manaf and Hassan [21] argue that the influence of S on fatty acid synthesis in plants should always be considered in conjunction with N supply. The small influence of N and S fertilization doses on fatty acid composition, as observed in the present study, allows an intensive use of these macronutrients, especially N, without fear of changing the fatty acid profile. This is particularly valuable in the case of agricultural varieties with altered fatty acid composition, as it is necessary to grow seeds of a certain quality. The results presented indicate that the fatty acid content in the oil of the winter oilseed rape genotypes studied was strongly influenced by the weather conditions—temperature and precipitation—during the stages of oil accumulation and seed maturation. This is in agreement with the results reported by other authors [21,52], that higher soil moisture and lower temperatures during seed development cause an increase in the content of linolenic acid (C18:3). The present study demonstrated that such weather conditions also contributed to an increase in linoleic acid (C18:2) content. In their study analysing cultivars with reduced linolenic acid (C18:3) content, Baux et al. [49] observed an increase in the content of this fatty acid at lower temperatures. Similar to other researchers [35,72], these authors showed an inverse relationship between the content of mono oleic (C18:1) and polyunsaturated linolenic acids (C18:3) with respect to temperature, and reported that the content of oleic acid (C18:1) increased at higher temperatures. Similar to the present study, Omidi et al. [73] showed that high temperature at maturity caused a significant increase in oleic acid (C18:1) and a decrease in linoleic acid (C18:2). Ahmad and Abdin [35] found large variations in oleic (C18:1) acid content during seed maturation. Wójtowicz [52,74] believes that the relatively higher content of oleic acid (C18:1) and the lower content of linolenic acid (C18:3) in the conditions of higher temperatures is the effect of the faster course of the seed ripening process at higher temperatures. Similar to the present study, Spasibionek et al. [54] demonstrated the effect of genotype–environment interaction in their study on new rapeseed genotypes, whose seeds accumulated oils with different stearic acid (C18) content. Thus, the significant influence of environmental parameters on the fatty acid composition of oil should be taken into account when planning rapeseed production.

4.3. Glucosinolate (GLS) Content in Seeds

The present study showed that the GLS content in the seeds of the winter oilseed rape genotypes studied was significantly influenced by the doses of both S and N. A significant increase in GLS content under the influence of N doses has also been demonstrated by other researchers [18,59,71]. Bilsborrow et al. [75] attributed this effect to an increase in N availability for GLS biosynthesis. Zhao et al. [59] showed that N fertilization can cause a change in the GLS profile, as a decrease in alkenyl GLS and an increase in indole GLS were observed under the influence of N. Groth [14] also found a decrease in the content of alkenyl GLS in winter oilseed rape as a result of increasing the N dose to 180 kg ha⁻¹. A slight but significant increase in the content of indole GLS was observed by Wielebski [30] when the N dose was increased from 60 to 120 kg ha⁻¹. Chen et al. [16] suggested that the accumulation of indole GLS could be enhanced by high doses of N and S. However, several studies have shown a non-significant effect of N fertilization on the content of these compounds [46,68,76]. Zhao et al. [38] suggested that the influence of N on GLS content in oilseed rape is determined by the availability of S and that, under conditions of S deficiency, N is mainly used for protein synthesis. Sulfur fertilization can deteriorate seed quality by increasing the GLS content [18,23,77]. It has been shown that, under the influence of S, total GLS concentrations in seeds of double-low winter oilseed rape varieties can increase in the range of 10% [17,78] to 80% [46,63]. The present study showed that S fertilization significantly increased alkenyl and total GLS content, while the difference in indole GLS content was insignificant. Some authors pointed out that the application of higher doses of S may cause a greater increase in the content of harmful alkenyl GLS compared to that of indole GLS [36,63], because S stimulates the synthesis of methionine, which is the precursor of alkenyl GLS [59]. A significant increase in the content of alkenyl GLS under the influence of S has also been confirmed by the results of several studies [14,34,43,44,76]. The influence of S on the content of GLS indoles is not clear, and is con-firmed by numerous reports in which, as in our own research, their contents were highly variable. [8,30,36,79]. Fismes et al. [6] found that balanced N and S fertilization allows better control of GLS content. The increase in GLS content in the seeds of new oilseed rape varieties due to S fertilization had no negative effect on the nutritional value of the seeds. The results of many studies have shown that winter oilseed rape shows differences in GLS content depending on soil moisture and thermal conditions [8,58]. Oleszek [56] found that GLS content was more influenced by water availability than by temperature. He suggested that this may be related to the availability and translocation of S, the uptake of which is limited under drought conditions, resulting in a reduction in GLS synthesis. In contrast, Jensen et al. [57] showed that drought increased GLS content. Similarly, the present study showed that in the years 2017 and 2018, when there was a lack of spring rainfall, the seeds were characterized by a significantly higher content of alkenyl GLS and a lower content of indole GLS. An inverse relationship was observed in the first year (2016), when there was sufficient spring rainfall. There was no significant difference in the total GLS content between the study years.

As outlined in this discussion, the importance of fertilization of oilseed rape with sulfur and nitrogen has been the subject of studies of many researchers. These have focused primarily on canola-type rapeseed, which does not contain erucic acid in oil and has a low content of undesirable glucosinolates. However, due to the use of rapeseed oil in different technologies, the breeding of varieties with new characteristics is developing. In particular, attention is being paid to oil stability, homogeneity, and suitability for deep frying. In response to these needs at the Plant Breeding and Acclimatization Institute, we have bred varieties with high oleic acid content (cv. Polka, genotype PN440) and a very low content of linolenic acid, responsible for rapid oxidation of rapeseed oil (genotype PN440). At the same time, these genotypes have the quality characteristics of the canola varieties, i.e., no erucic acid in the oil and low glucosinolate content in the seeds. The introduction of new types of cultivars into farming practice requires knowledge of their cultivation requirements, particularly of the fertilization, with the main elements determining the quality and quantity of oilseed rape yield. Our studies have shown that genotypes with changed fatty acid profiles react similarly to changes in environmental conditions, as well as fertilization, like other canola cultivated varieties. It is important to note that the applied fertilization with nitrogen and sulfur and the different environmental conditions did not have a highly significant effect on the quality of the new oilseed rape genotypes tested. In the future, it will be good to perform this type of study on a larger number of similar genotypes.

5. Conclusions

The studies carried out showed that the doses of nitrogen (N) applied (100, 160 and 220 kg ha⁻¹) caused a decrease in crude fat content and an increase in protein content in seeds of all types of investigated winter rapeseed genotypes. The low and medium doses of N (100 and 160 kg ha⁻¹) and the low dose of sulfur (S) fertilization (30 kg ha⁻¹) were associated with a significant increase in crude fat content and a decrease in protein content.

An interaction between N and S has been noted, indicating that there is a requirement to balance the application of these nutrients for the optimum quality of parameters in seeds.

The application of S and N poorly differentiated the composition of fatty acids, which was particularly beneficial for the HO and HOLL types of winter oilseed rape with changed fatty acid composition.

The studies have shown that different levels of N and S significantly influenced the content of glucosinolates (GLSs) in the seeds of three winter oilseed rape genotypes. Increasing the N dose from 100 to 160 kg N ha⁻¹ caused a significant increase in the sum of alkenyl and total glucosinolates (GLSs).

The seed- and oil-quality traits studied were significantly influenced by weather conditions—temperature and rainfall.

These results are relevant because oilseed rape is one of the major sources of edible oil in the world, second after palm oil. Breeding efforts continue to focus on improving the quality and quantity of oil in rapeseed seeds to ensure wide use of this oil in the food industry and other technologies. Examples are the HO and HOLL varieties used in the research presented here. A by-product of rapeseed oil extraction is post-extraction meal containing protein similar in quality to soybean meal. Rapeseed protein is primarily used for animal feed, but has the potential to be directly consumed in food products. Due to the growing population, research is needed to ensure food security. Research into the optimization of rapeseed production in terms of quantity and quality is essential.

A better knowledge of the interactions between N and S in relation to seed quality could improve fertilizer management in a sustainable way. This study suggests that limiting fertilizer inputs while maintaining or even improving oil quality and seed meal may become an important environmental and economic priority.