Exploring Polyploidization in Nigella sativa L.: An Applicable Strategy Towards Crop Improvement

Abstract

A plant breeding program needs helpful markers, especially morphological ones, which can allow breeders to dispense with other markers, including cytological traits and flow cytometry. These markers can assist plant breeders in distinguishing diploid and tetraploid plants during the seedling stage. Therefore, the present study aimed to investigate and validate effective methodologies for the early identification of artificially induced polyploids in black cumin. Thus, we established an extensional program for black cumin breeding including producing seeds, active compounds, and flowers as ornamental plants. Field experiments on tetraploids and diploids were carried out to evaluate the morphological and yield traits of both plants. Also, some cytological studies and Gas Chromatography (GC) analysis were conducted to achieve these goals. The results showed the possibility of realizing diploid and tetraploid plants in early growing black seeds in the field (mainly after the first cotyledon leaves). This crucial outcome can support plant breeders in identifying polyploidy during the seedling stage without referring to biochemical markers, flow cytometry, and cytological traits. All morphological and yield-related traits were superior in diploid plants compared to tetraploids. The results showed that diploid and tetraploid plants exhibited plant heights of 116 cm and 95 cm, numbers of secondary branches of 112 and 22, numbers of flowers of 111.7 and 24.75, and shoot fresh weights of 610 g and 147.5 g, respectively. Furthermore, the number of seeds per capsule, seed yield per plant, and oil percentage in diploids were 97.5 seeds, 24 g, and 22.94%, compared with 35.25 seeds, 4.62 g, and 17.76% in tetraploids, respectively. This work used the cotyledon leaf shape as a morphological marker to distinguish the tetraploid and diploid plants, as diploids are typically taller with pointed cotyledons, whereas tetraploids are shorter with rounded cotyledon tips. This study will create great opportunities for plant breeders to save time and costs during their programs. Further studies on such suggested black cumin breeding programs are needed on diploids, triploids, and tetraploids.

1. Introduction

Nigella sativa L., commonly known as black cumin or black seed, is an annual herbaceous plant belonging to the family Ranunculaceae. It has a diploid chromosome number of 2n = 2x = 12. This plant species has several culinary uses and has been recognized in traditional medicine for treating many ailments such as eczema, headache, inflammation, anorexia, rheumatism, and paralysis [1,2,3,4]. It has been widely used in different types of product such as essential oil, extract, powder, and paste [5,6]. It is native to Southern Europe, Southwest Asia, and North Africa. It is cultured in the Middle East, the Mediterranean, and Southern European regions [7]. Black cumin seeds are often used as food preservatives, spices, and flavoring additives for adding a distinctive aroma and taste to bread, savory dishes, and pickles due to their low level of toxicity [8,9]. Moreover, the seeds are known for their nutritional value due to their high content of iron, copper, phosphorus, calcium, zinc, folic acid, thiamin, niacin, and pyridoxine [10,11]. The pharmaceutical and commercial value of black cumin is determined by its seed yield, essential oil content, and concentration of bioactive compounds [12]. Due to the health benefits of black cumin, it is necessary to develop plants with desirable traits through induced genetic variations and breeding programs, as the existing germplasm may not satisfy future demands [12].

Polyploidy or whole genome duplication is the heritable state in which the cells carry more than two complete chromosomal sets [13]. The somatic cells of polyploid organs have more than two complete/paired sets of homologous chromosomes [14]. Polyploidy, or the doubling of the chromosome number, is one of the plant breeding methods for genetic improvement of plants. Autotetraploid induction for plants has been studied for over 85 years [15]. Tetraploidy of black cumin was chemically induced by colchicine treatment of different plant organs, i.e., seed and seedling [16], seedling [17], and apical meristematic tips of young seedlings bearing only two cotyledonary leaves [18], or by another method that involves using 2,4-dinitroaniline treatment on seeds [19].

Previous reports indicated that polyploidy extended flowering duration, increased organ size, and improved resistance to abiotic stresses and diseases [20]. For black cumin, induced tetraploid plants using colchicine had more branches and flowers, enhanced size and frequency of stomata, and an increased number of septa per fruit and seeds per septum [16]. Furthermore, leaf thickness, flower and anther size, and petals were increased in tetraploid black cumin plants [21]. Autotetraploid induction improved the fruit quality of kiwifruit (Actinidia chinensis Planch.) in weight, size, and diameter [22]. Tetraploids of the Anemone sylvestris L. flowering plant are characterized by strong, vigorous growth, and they flower early compared to diploid plants [23]. In addition, artificial polyploidy enhanced the biosynthesis of important secondary metabolites in many polyploidy plants compared to their diploid parents [24]. Tetraploid clones of Artemisia annua L. (synonym: Artemisia chamomilla C.Winkl.) increased artemisinin production six-fold compared to the diploid parent [25]. Tetraploid plants of radish (Raphanas sativus L.) have larger vegetative and reproductive organs, enhanced antioxidant enzyme activity, and higher soluble contents and quality [26]. One autotetraploid black cumin plant had some useful traits compared to diploids and other tetraploids [18]. Polyploidy induction enhances plant breeding potential by improving ornamental traits, stress tolerance, yield, and fertility restoration, and enabling the development of seedless cultivars.

On the other hand, induction of artificial polyploidy can negatively affect plant growth and fruit size for commercial production [27]. The growth of diploid plants of apple was more vigorous than tetraploid ones, and the stomatal conductance and photosynthetic rate of tetraploid plants were slightly lower [28]. Vegetative growth and yield of diploid black cumin plants were higher than tetraploid plants [21]. Seeds of tetraploid plants of black cumin were sterile due to widespread chromosome irregularities, producing varying numbers of quadrivalents (0–4), trivalents (0–2), and univalents (0–10) [17]. The most prominent morphological changes in black cumin tetraploid and its progenies were increased flower and capsule sterility, and reduced seed number per capsule and seed fertility [18].

Early identification of polyploids is a crucial issue in developing plant breeding programs. It was reported that approximately 70% of all angiosperms have undergone one or more polyploidy events [29]. One common method for identifying polyploids is morphological based-markers like size, shape, and color of plant organs (e.g., cotyledons, leaves, and flowers). It is a practical method for distinguishing polyploid plants, especially at the early stages of growth. Tetraploid radish seedlings were significantly taller (by 36%), had longer (61%) and wider (43%) leaves, and exhibited larger floral organs as compared to diploid plants [26]. Similarly, notable differences in plant height, leaf length, petiole characteristics, and leaflet width were observed between diploid and tetraploid plants [30]. Physiological markers are also considered as a reliable tool for polyploid identification. These include stomatal characteristics such as size, density, and number of chloroplasts per guard cell. It was demonstrated that stomatal size tends to increase in polyploid plants, while stomatal density generally decreases, providing a novel method for detecting polyploidy [29]. Later studies confirmed that polyploidy leads to increased stomatal size and chloroplast number, accompanied by reduced stomatal density [26,31,32]. Another precise method is chromosome counting during mitotic or meiotic division. Tetraploidy was confirmed in radish by counting chromosomes in pollen mother cells at metaphase I during meiosis [26]. Similarly, tetraploidy in black cumin was verified through mitotic chromosome analysis [18,19]. Flow cytometry is also widely used for determining ploidy levels by comparing nuclear DNA content between diploid and tetraploid plants. This technique has been successfully applied in various species, including radish [26], black cumin [33], purple coneflower [34], and Urochloa [35].

A limited number of studies have addressed the comparative evaluation of diploid and tetraploid black cumin, primarily focusing on mature plants. These studies have relied on various approaches, including morphological and physiological measurements, cytological assessments, and flow cytometry analysis [17,18,19,33]. However, these studies provide little information on early developmental stages, which are critical for accelerating selection processes in breeding programs. In the present study, we extend the scope of evaluation by comparing diploid and tetraploid black cumin at both the early cotyledon stage and the mature plant stage. This dual-stage approach allows for the identification of early morphological and physiological indicators that can reliably distinguish polyploid plants. Such early detection methods are particularly valuable for plant breeders, as they reduce the time and resources required to confirm ploidy status at later growth stages. The main objective of this study was to explore and validate practical methodologies for the early identification of artificially induced polyploids in black cumin. In addition, this study sought to assess economic traits such as plant vigor, leaf and flower morphology, potential yield components, and thymoquinone (TQ) content of diploid versus tetraploid plants in order to evaluate their agronomic significance.

2. Materials and Methods

This research was performed in 2021–2022 at the Physiology and Breeding of Horticultural Crops Laboratory, Horticulture Department, Faculty of Agriculture, Kafrelsheikh University, Egypt.

2.1. Plant Materials

In the present study, seeds from a tetraploid breeder line (induced using 2,4-dinitroaniline at 10 mg L⁻¹ for 24 h) and a selected diploid line obtained from the breeding program (El-Mahrouk et al. [19]) at the Faculty of Agriculture, Kafrelsheikh University, Egypt, were obtained, identified, and compared.

2.2. Cytological and Flow Cytometry Analysis

Flower buds of diploid and tetraploid plants were treated with freshly prepared fixation solution (three parts absolute alcohol/one part glacial acetic acid) for 24 h. Samples were preserved in 70% ethanol and placed in a cool place for scanning. Anthers were excised on the slide and stained with acetocarmin, then sealed and observed under a Leica Aristoplan light microscope (Neu-Isenburg, Germany) with Leica DC 300 F digital imaging using an oil immersion lens [36]. Ploidy level was detected by an Attune flow cytometer (Applied Biosystem, Foster City, CA, USA). Young leaves from seedlings (30 days old) of diploid and tetraploid plants were used as samples according to Galbraith et al. [37]. Leaf tissue (50 mg) was chopped and macerated in lysis buffer (1.0 mL) to release intact nuclei by a razor blade in Galbraith buffer [45 mM MgCl₂; 30 mM sodium citrate; 20 mM MOPS [3-(N-morpholino)-propane sulfonic acid]; 0.1% (w/v) Triton X-100; pH 7.0] for 1 h. The cell suspensions were filtered by a 0.45 µm nylon filter to eliminate cell debris for 5 min at room temperature. Then, the cell nuclei were stained with 10 µL 4′,6′-diamino-2phenylidole (DAPI) solution (solution A of high-resolution kit type P, Partec) for 30 min on ice in the dark. Nearly 10,000 nuclei were analyzed using a logarithmic scale. Histograms were analyzed using Attune cytometric software version 2.1.

2.3. Establishment of Seedlings

Seeds of tetraploid plants were cultivated separately in isolated field to avoid cross pollination with other black cumin plants to maintain pure tetraploid seeds. Under greenhouse conditions, the seeds were germinated in peat moss and vermiculite mixture (1:1; v/v). The medium was fertilized with 1.0 g L⁻¹ of water-soluble compound fertilizer in a ratio of 19:19:19 (Rosasol; Rosier, Moustier, Belgium) and sterilized with a solution of fungicide (1.0 g L⁻¹ Rizolex; Kafr El-Zayat Company, El-Gharbia, Egypt). The pH of the medium was adjusted to 6.0 ± 1 with calcium carbonate (Jenway 3510, Staffordshire, UK). Soaked seeds (for 24 h in distilled water) were sown in expanded polyurethane foam trays (3.0 × 3.0 cm; 209 cells/tray), with one seed in each cell and one for each diploid and tetraploid line. Trays were put in the greenhouse at 25 ± 2 °C and light intensity of 300 μm m⁻² s⁻¹ after sowing. The trays were also manually watered weekly using 10 L watering cans with 2.0 L for each tray. All trays were covered with plastic sheets (35 µm) until the first germinated seeds became visible, and then the plastic covers were removed. Germination indices including final germination percentage (FGP), germination rate index (GRI), corrected germination rate index (CGRI), and time to 50% germination (GT50) were recorded. At this stage, the morphological shape of cotyledons and chromosomal number in mitosis division were recorded. The seeds were considered germinated when their cotyledons became visible above the surface of the growth medium. Germination parameters were calculated as follows:

$$\begin{array}{c}\left(\mathrm{a}\right) \mathrm{Final} \mathrm{germination} \mathrm{percentage} \left(\mathrm{FGP}\right)\\ =[\mathrm{Number} \mathrm{of} \mathrm{germinated} \mathrm{seeds} \mathrm{after} 30 \mathrm{days} \mathrm{from} \mathrm{sowing} \\ /\mathrm{number} \mathrm{of} \mathrm{sown} \mathrm{seeds}] \times 100\end{array}$$

$$\left(\mathrm{b}\right) \mathrm{Germination} \mathrm{rate} \mathrm{index} \left(\mathrm{G}\mathrm{R}\mathrm{I}\right) = \left[\left({\displaystyle \frac{\mathrm{G}1}{1}}\right)+\left({\displaystyle \frac{\mathrm{G}2}{2}}\right)+\left({\displaystyle \frac{\mathrm{G}\mathrm{x}}{\mathrm{X}}}\right)\right]$$

where G = germination on each alternate day after placement; 1, 2, x = corresponding day of germination [38,39].

$$\left(\mathrm{c}\right) \mathrm{C}\mathrm{o}\mathrm{r}\mathrm{r}\mathrm{e}\mathrm{c}\mathrm{t}\mathrm{e}\mathrm{d} \mathrm{g}\mathrm{e}\mathrm{r}\mathrm{m}\mathrm{i}\mathrm{n}\mathrm{a}\mathrm{t}\mathrm{i}\mathrm{o}\mathrm{n} \mathrm{r}\mathrm{a}\mathrm{t}\mathrm{e} \mathrm{i}\mathrm{n}\mathrm{d}\mathrm{e}\mathrm{x} \left(\mathrm{C}\mathrm{G}\mathrm{R}\mathrm{I}\right)=\left({\displaystyle \frac{\mathrm{G}\mathrm{R}\mathrm{I}}{\mathrm{F}\mathrm{G}\mathrm{P}}}\right)\times 100$$

$$\left(\mathrm{d}\right) \mathrm{G}\mathrm{T}50=\mathrm{n}\mathrm{u}\mathrm{m}\mathrm{b}\mathrm{e}\mathrm{r} \mathrm{o}\mathrm{f} \mathrm{d}\mathrm{a}\mathrm{y}\mathrm{s} \mathrm{l}\mathrm{a}\mathrm{p}\mathrm{s}\mathrm{e}\mathrm{d} \mathrm{t}\mathrm{o} \mathrm{r}\mathrm{e}\mathrm{a}\mathrm{c}\mathrm{h} 50\mathrm{\%} \mathrm{o}\mathrm{f} \mathrm{F}\mathrm{G}\mathrm{P}$$

2.4. Soil Analysis and Cultivation of Black Cumin Seedlings

The soil analysis of the experimental site is presented in

Table 1. Soil chemical analysis of experimental farm of the Horticulture Department, Faculty of Agriculture, Kafrelsheikh University.

EC (dS m−1) (1:5)	pH (1:2.5)	Available Nutrients (mg kg−1)			Soluble Cations (mmolc L−1)				Soluble Anions (mmolc L−1)				OM (%)
		N	P	K	Mg2+	Ca2+	K+	Na+	CO32−	HCO3−	Cl−	SO42−
3.96	8.13	32.65	10.9	380	7.1	12.47	0.24	19.77	0.0	3.33	18.4	17.9	1.45

. Soil pH was measured in a 1:2.5 ratio (soil/deionized water suspension) using a calibrated pH meter 3510 (Jenway, Stafford Shire, UK). Soil salinity [electrical conductivity (EC)] was measured in a 1:5 ratio (soil/deionized water) using an EC Meter (MI 170; Treviglio, Italy). Soluble ions in saturated extracts were measured according to the methods of Jackson [40]. Total carbonate was determined using a volumetric calcimeter [41]. Organic matter content was determined using the dichromate oxidation method [41]. Available nitrogen (NH₄⁺) was determined using the micro Kjeldahl method [42], and available phosphorus (P₂O₅) was determined [43]. Calcium (Ca²⁺) and magnesium (Mg²⁺) were also measured [40]. Sodium (Na⁺) and potassium (K⁺) were extracted according to the methods described by Black [44], and concentrations were determined using a Flame photometer PFP7 (Jenway, Stafford Shire, UK). Chloride (Cl⁻) was determined by titration with a standard solution of silver nitrate [40]. Both diploid and tetraploid seedlings of black cumin were transplanted in clay soil during two successive seasons, 2021 and 2022, in the experimental farm of the Horticulture Department, Faculty of Agriculture, Kafrelsheikh University. Each genotype was planted in five rows (6.0 × 0.8 m), each row considered as one replicate. The distance between plants was 30 cm. The experiment was maintained for 7 months. All cultural practices for best plant growth were followed according to Rajeswara et al. [45].

2.5. Assessment of Plant Morphology, Flowering, and Yield Traits Under Field Conditions

The morphological traits of tetraploid plants and their corresponding diploid plants growing under the same field conditions were employed to verify the polyploidy variations. Ten seedlings from each genotype were used for determining the shape and area of cotyledon leaves. Twenty-five plants from every genotype were randomly selected from five replicates in order to assess the following traits. Morphological vegetative traits including plant height, number of main and secondary branches per plant, crown diameter (cm), shoot fresh and dry weights, root length (cm), and root fresh weight. Morphological flower traits in terms of the number of first days to flowering, number of flowers per plant, converted flowering parts, and stamen number and size were also investigated, as well as yield traits in terms of the number of seeds per capsule, seed yield per plant, and 100-seed weight. Oil percentage was determined according to Folch et al. [46].

2.6. Analysis of Thymoquinone Content

An analysis of thymoquinone content in the seed extracts was carried out in the High Institute for Public Health Central Lab, Alexandria University, using the Gas Chromatography GC-HP (Hewlett Packard) 6890, with FID detector (flame ionizing) and DB-23 Column (50% cyanopropyl–methylpolysiloxane), 30 m × 0.32 mm, ID = 0.25 μm film thickness, to determine TQ content. The carrier gas was nitrogen (1 mL min⁻¹ gas flow). The seed oil of diploid and tetraploid plants (extracted from 9 seeds) was dissolved in HPLC methanol to prepare them for GC analysis. Samples of 1.0 μL were injected at a temperature of 220 °C. The oven temperature for the first 2 min was 100 °C and increased at 10 °C min⁻¹ until 240 °C, then was held for 2 min. Injector and detector temperatures were set at 250 °C. Thymoquinone standard (2-isopropyl-5-methyl-l, 4 benzoquinone) 99% (Sigma Chemical Co. St Louis, MO, USA) was dissolved in HPLC methanol, whose dilution factor was 19.7 mg 0.2 mL⁻¹ methanol. The concentration of thymoquinone of the seeds was calculated using the following equations:

$$\begin{array}{c}TQ concentration (\mathrm{m}\mathrm{g}/\mathrm{m}\mathrm{L})\\ = \left(\right(area of sample\\ ÷ area of standard) 98.5) \times dilution factor)\\ /volume of used extract\end{array}$$

$$T\mathrm{Q} \mathrm{c}\mathrm{o}\mathrm{n}\mathrm{s}\mathrm{e}\mathrm{n}\mathrm{t}\mathrm{r}\mathrm{a}\mathrm{t}\mathrm{i}\mathrm{o}\mathrm{n} \mathrm{o}\mathrm{f} 1 \mathrm{g} =\mathrm{T}\mathrm{Q} \mathrm{c}\mathrm{o}\mathrm{n}\mathrm{c}\mathrm{e}\mathrm{n}\mathrm{t}\mathrm{r}\mathrm{a}\mathrm{t}\mathrm{i}\mathrm{o}\mathrm{n} \left(\mathrm{m}\mathrm{g}/\mathrm{m}\mathrm{L}\right) \times \mathrm{n}$$

n number of miles extracted from 1 g plant material.

$$TQ concentration (\mathrm{m}\mathrm{g}/100 \mathrm{g} plant material)=TQ concentration of 1 \mathrm{g}\times 1000$$

2.7. Statistical Analyses

The experiments were set up in a completely randomized block design. Data were analyzed with one-way ANOVA and the unpaired t-test in SAS software (version 9.4; SAS Institute, Inc., Cary, NC, USA).

3. Results

3.1. Cytological and Flow Cytometry Analysis of Diploid vs. Tetraploid Plants

Concerning the cytological analysis, the normal meiosis for diploids (n = x = 6) and tetraploids (n = 2x = 12) of black cumin plants is presented in Figure 1. Due to its potential as an important proposed method for ploidy analysis of different polyploidy levels after applying 2,4-dinitroaniline, flow cytometry was used to confirm the production of tetraploid through treatment. The flow cytometry data are displayed as a histogram of relative fluorescence intensity, representing relative DNA content. The histogram peak of mean fluorescence intensity (MFI) was 4495 and 10,520 for diploid and tetraploid plants, respectively. By accounting for the total area under the curve or MFI, there seems to be a higher total area for the tetraploid plants compared with the diploids.

3.2. Germination of Diploid and Tetraploid Black Cumin

The effect of ploidy level on germination parameters including final germination percentage (FGP), germination rate index (GRI), corrected germination rate index (CGRI), and median germination time (GT₅₀) in black cumin is presented in Figure 2. The data showed that germination started for diploid and tetraploid soaked seeds after 4 and 8 days, respectively. All these measured parameters, including FGP, GRI, and CGRI, showed a higher germination rate for the diploids of the studied plants compared to the tetraploids, except GT50 Days. The increasing rate of germination parameters was recorded at 125, 66.6, and 60% for FGP, GRI, and CGRI, respectively, in the diploid plant, whereas the GT50 was 8.9 and 14.2 days for the diploid and tetraploid plants, respectively. Also, the polyploidy level affected the number of days for germination of 50% of the seeds (GT50), and the positive effect was for the diploid plant rather than the tetraploid plant. Significant changes could also be observed in all the studied germination parameters between the diploid and tetraploid plants.

3.3. Morphology of Black Cumin Seedlings in Diploid Versus Tetraploid Plants

Various morphological traits of both diploid and tetraploid plants are presented in

Table 2. Key morphological traits of diploid and tetraploid black cumin plants.

Trait (Unit)	Diploid	Tetraploid	p-Value
Plant height (cm)	116.50 ± 0.866	95.00 ± 2.887	0.001021 *
The main number of branches	10.50 ± 0.289	8.75 ± 0.144	0.002804 *
Number of secondary branches per plant	112.00 ± 2.309	22.00 ± 1.155	0.00001 *
First days to flowering (day)	132.70 ± 1.443	97.09 ± 3.453	0.00001 *
Number of flowers per plant	111.70 ± 1.732	24.75 ± 0.289	0.00001 *
Flower diameter (cm)	3.70 ± 0.058	3.27 ± 0.010	0.000918 *
Converted flowering parts (petaloid stamen)	20.34 ± 0.510	188.00 ± 1.020	0.00001 *
Stamen number	43.34 ± 0.820	9.44 ± 0.290	0.00021 *
Capsule diameter (cm)	1.49 ± 0.078	0.83 ± 0.880	0.002601 *
Shoot fresh weight (g)	610.00 ± 5.774	147.50 ± 1.443	0.00001 *
Shoot dry weight (g)	110.50 ± 2.598	37.00 ± 1.732	0.00001 *
Crown diameter (cm)	1.91 ± 0.052	1.16 ± 0.118	0.002142 *
Leaf area (mm)	17.50 ± 0.058	22.50 ± 0.289	0.000035 *
Root length (cm)	21.65 ± 0.202	19.00 ± 0.577	0.006164 *
Root fresh weight (g)	8.00 ± 0.577	5.95 ± 0.029	0.011939 *

* = significant at p < 0.05 according to Student’s unpaired t-test.

. All measured morphological characteristics were higher in diploid plants than in tetraploids, except for converted flowering parts and leaf areas, which were greater in tetraploids (188 cm and 22.5 cm, respectively) compared to diploids (20.34 cm and 17.5 cm, respectively). The analyzed traits included plant height, number of main and secondary branches per plant, days to first flowering, number of flowers per plant, stamen number, shoot fresh and dry weight, crown diameter, root length, and root fresh weight. The results indicated that most of these traits were several times higher in diploids than in tetraploids, ranging from three- to ninefold differences, particularly in stamen number, number of flowers per plant, and shoot fresh and dry weight.

Clear differences in leaf morphological features were observed between diploid and tetraploid plants, including variations in cotyledon and mature leaf shape (Figure 3a–f). The cotyledon leaf of a diploid is taller and more pointed than the shorter tetraploid, which has rounded leaf tops, whereas the mature leaves are lower in thickness and more trimmed than tetraploid ones. On the level of field plants, the plant size of the diploid plants at the flowering stage of both diploid and tetraploid plants was observed and considered. It was observed that diploid plants exhibited greater growth and branch intensity compared to tetraploid plants. The flower shapes of diploid and tetraploid plants and stamen size were compared. This is a morphological analysis of diploid and tetraploid plants performed by examining the impact of applied 2,4-dinitroanaline on the plant polyploidy. It is apparent that the flower size of the tetraploid plants was bigger than that of the diploid plants (Figure 4a–d), and the same trend for stamen size can be noticed (Figure 4e). Also, it is observed in tetraploid plants that a number of stamens converted to petals, affecting the flower size and fertility.

3.4. Yield Traits and Thymoquinone Content in Diploid vs. Tetraploid Plants

Figure 5 shows the yield traits of the diploid and tetraploid black cumin plants (i.e., number of seeds per capsule, seed yield per plant, 100-seed weight, and oil percent). All previous yield parameters recorded higher values for the diploid plants compared with the tetraploid plants except the 100-seed weight. The number of seeds per capsule, seed yield per plant, 100-seed weight, and oil percent were 96.5, 24 g, 0.205 g, and 22.5% of the diploid plants, respectively, and 36.5, 4.5 g, 0.385 g, and 17.67% of the tetraploid plants, respectively. Thus, the 100-seed weight of the tetraploid plants was double that of the diploid plants. Data presented in Figure 6 show the thymoquinone percentage in the diploid and tetraploid plants. The obtained results indicated that tetraploid plants are superior to diploid plants in thymoquinone content, with 13.58 and 11.45 mg/100 g seeds, respectively (Figure 6D).

4. Discussion

Chromosome counting and flow cytometry could be used for genotyping-based ploidy determination in most accessions of Yams (Dioscorea spp.), allowing future rapid screening of ploidy levels [47]. Flow cytometry measures the relative fluorescence intensity that refers to the relative DNA content or genome size in the nuclei of the polyploidy samples compared to diploids. In our results the MFI of diploid plants was nearly half that of tetraploid. So this technique is widely used as the most convenient and rapid method for screening DNA ploidy level in plants [33,48,49]. Previous studies reported that flow cytometry has much higher accuracy than any other methodology for measuring ploidy, so it has become the best assay for ploidy level detection [50,51]. Therefore, diploid and tetraploid plants could be confirmed using flow cytometry assessment.

Ploidy level in black cumin was significantly associated with variations in seed germination. Diploid plants exhibited a higher germination percentage and required fewer days to reach 50% of final germination percentage (FGP) compared to tetraploid plants. Polyploidy may influence seed establishment, germination, and development [52]. Also, interactions between polyploidy level and the seed developmental environment may affect subsequent dormancy, and early growth traits, particularly under stress conditions [53]. A previous study indicated that tetraploid seeds had higher dormancy and a lower germination rate as compared with diploid seeds [53,54].

The present study highlights three morphological markers that can be effectively applied in black cumin breeding programs. Notably, cotyledon leaf shape proved to be a reliable indicator of ploidy level, as diploids consistently exhibited pointed tips, whereas tetraploids displayed rounded tips. These findings align with previous reports demonstrating that cotyledon morphology in black cumin is influenced by ploidy level, thereby supporting the use of this trait as an early, practical marker for distinguishing cytotypes [4]. This important feature can help black cumin breeders to detect the polyploidy level, and select tetraploid plants in the early growing stage, saving time and costs. The second marker was a little leaf-slitting and thicker leaves for the tetraploids, whereas the opposite was observed for the diploids. Previous studies demonstrated that triploids and tetraploids showed a wide range of variations in morphological traits, such as thicker and darker-green leaves [4,55]. The third marker was the flowers; there are obvious differences between diploids and tetraploids. These differences are due to the stamen number, which was more than fourfold for the diploids (43.34) what it was for the tetraploids (9.44). A possible explanation for using tetraploid black cumin plants in a breeding program as ornamental plants might be due to their converting stamens into petals, producing a big flower and increased longevity of the flowers. These findings for tetraploids are in agreement with the earlier flowering (after 97 days), lower number of flowers (24 per plant), and converted flowering parts (188), which increased the size of flowers. Another possible explanation for this result may be the increased longevity of these flowers, and the infertile flowers may be due to converting flowering parts into petaloid stamen.

Morphological traits of black cumin are crucial parameters, as they are closely linked to yield components and essential oil production. Tetraploid black cumin plants exhibit distinct morphological changes under field conditions. Several previous studies confirmed the potential value of black cumin traits (e.g., [56,57]). The differentiation in morphological characteristics between the diploid and tetraploid plants was investigated in both seedlings and mature plants. The start of flowering was earlier in January for tetraploids, whereas it was in February for diploids. The most interesting finding was that only the 100-seed weight of tetraploid plants was higher than that of diploid plants. The same finding was observed by Stevens et al. [53] on perennial grass. They found that tetraploid seeds were significantly heavier than diploid seeds by an average of 35%, whereas the yield of seeds associated with diploid plants was recorded as being fivefold that of tetraploids. A possible explanation for tetraploids’ lower seed yield may be the conversion of flowering parts into petaloid stamen (infertile flowers). Another possible explanation for this result may be the big thickness of pollen tubes in tetraploids, which slow growth in the stigma tissues and then delay the fertilization of ovules, lowering the number of seeds in each ovary. Tetraploid plants exhibited stalk-lodging at maturity and low levels of segregation in the F2 and upcoming generations [55]. Tetraploids of Jatropha curcas L. possessed a decreased number of flowers per inflorescence, fruits per fluorescence, and seeds per fruit. This condition is due to lower fertility of pollen of the tetraploid plants and/or the slow rate of the photosynthesis process [58]. Tetraploids of Echinacea purpurea (L.) Moench exhibited larger flowers and seeds [34], and significantly larger pollen grains were noticed in tetraploids of Ocimum basilicum L. [59]. On the other hand, black cumin producers can benefit from the tetraploid genotype by intensifying planting to obtain a high yield near the diploid plant.

Although the polyploidy may improve some traits, sometimes it can affect others negatively. In this context, flowering and bolting genes of radish were reported to have lower expression in tetraploid compared to diploid plants; however, tetraploid plants were greater in size in terms of both vegetative and reproductive organs [26]. Tetraploidy induction (2n = 4x = 24) was reported on wallflower (Erysimum cheiri (L.) Crantz), which enhanced the morphological, physiological, and biochemical traits of this plant [60]. In our present study, it was found that the tetraploid plants of black cumin had a negative effect on the traits related to vegetative and yield components. A possible explanation for these results may be the tetraploids, which often can alter rates of vegetative growth relative to their diploid progenitors. The degradation of chloroplast in tetraploid leaves was accelerated, the rate of photosynthesis was decreased, and then the synthesis of carbohydrates was decreased [61]. Previous studies indicated that polyploid plants have slower growth and development [62], because the difficulties happen in the cell cycle in addition to slow cell division [63], which resulted in decreased cell numbers and smaller organs.

A program of plant breeding of a certain crop like medicinal plants of promising value has many strategies depending on its purpose. A program for cumin plants is preferable to using tetraploids for producing high essential oil compared to diploids [64]. The producing tetraploids of some plants have many desirable characteristics compared to diploid plants, such as potato tubers of big size [65,66]. Black cumin crops can be propagated for three purposes: producing seeds, a high content of active compounds in oil, and the desired flowers. Therefore, the breeders of black cumin production face many challenges depending on the purpose of such production, which may include (1) producing the seeds by propagation of diploid plants, (2) producing the active compounds through a callus of tetraploids, and (3) producing the flowers, as ornamental plants, via triploid and tetraploid plants.

Producing active compounds (mainly thymoquinone) in black cumin seeds is a crucial issue for the breeders of this crop. This production can be achieved monthly through the callus protocol of tetraploids [7]. In Egypt, the production of black seeds is mainly in May, and postharvest, the seeds should be under cooling and modified conditions to keep such compounds active and ready for cultivation after storing for one year. In the current study, the 100-seed weight was reported to be high in tetraploids compared to diploid plants of black cumin, but at the same time, these findings suggest the lowering seed yield of tetraploids, as mentioned before. The oil percent in black seeds was higher in diploids than in tetraploid plants, along with the yield of seeds, which has the same trend. The variability in active compounds content in different varieties of the plant grown in different regions has been studied [67,68]. The significant differences in the environmental conditions at various sites, and genetic variation of the plant material, are what affect the phytochemical content. Additionally, advanced analytical methods enabled us to explore black cumin’s environmental, genotypic, and ontogenetic variability regarding its thymoquinone content [69]. Thymoquinone in black cumin depends on the plant’s genetic and environmental conditions. Our results indicated that tetraploid plants had more concentration of thymoquinone than diploid plants. The same finding was reported for tetraploid lines of black cumin where they contained higher concentrations of thymoquinone in the seeds’ oil by a ratio of approximately 46% [55]. Several studies assumed that synthetic polyploidization might enhance the biosynthesis of primary and secondary metabolites due to chromosome duplication that could affect the biological activities of the polyploid plants [70,71,72].

We compared in our published studies the production of diploid and tetraploid plants of black cumin and their comparison at the mature stage under field conditions (e.g., [19,21,33]). In contrast, this study is the first report on such a comparison at the early growth stage (seedling). The findings of the current research on tetraploid plants confirmed that these plants are a treasure for black cumin breeders because of their superiority in one or more of their genotypes. Therefore, it is necessary to identify them in their different growing stages and by several ways. The most obvious finding from the current investigation is that the differentiation between diploid and tetraploid plants could be distinguished during the seedling stage.

The production story began in 2015 with El-Mahrouk et al. [19], followed by cultivation of tetraploid plants for more than 5 years until 2021 to confirm this production of tetraploid plants. During the two successive seasons, the tetraploid and diploid seeds were sown while recording important guidelines for the studied breeding program. The first result is distinguishing between diploids and tetraploid seeds after germination under field conditions. The second one clarifies the suggested three strategies of black cumin production, including producing seeds, active compounds, and flowers of this plant. The third one is how to orient the plant breeding of black cumin by focusing on the morphological and cytological attributes and selecting the right time for cultivating practices from germination to harvesting. This study opens considerable opportunities for more research on black cumin plant breeding programs.

5. Conclusions

Black cumin is considered a highly potent medicinal plant with unique active phytochemicals for its therapeutic potential, and several benefits. Black cumin crops can be mainly propagated for three purposes (producing seeds, high content of active compounds in oil, and the desired flowers). The present study provides an important reference for black cumin breeders. Cotyledons can be an excellent marker for distinguishing between tetraploid and diploid plants due to the possibility of identifying polyploid plants at an early stage of growth (cotyledon stage). This approach can help breeders to avoid undesirable hybridizations that result in sterile plants without any crop and to determine the intended purpose of black cumin cultivation. Furthermore, vegetative trait markers could help in reducing the high cost of genetic markers or flow cytometry. Our study showed that diploid plants are superior in most morphological traits (i.e., plant height; number of branches, flowers, and stamens; and vegetative fresh weight) and yield traits (i.e., number of seeds per capsule, seed weight per plant, and oil percentage) when compared with tetraploid plants. Despite the negative effects of polyploidy in black cumin on both vegetative and yield traits, it had a positive and promotional effect on the ratio of thymoquinone content and 100-seed weight. This may indicate the possibility of producing tetraploid plants commercially through intensive planting. This study contributes valuable information to ongoing efforts in black cumin improvement. The findings support the integration of morphological markers as a cost-effective and accessible tool in polyploid breeding programs aimed at enhancing the medicinal and economic value of this important species.