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26 February 2026

21 Pages

Drought Stress Response of Doubled Haploid Interspecific Rapeseed Hybrids at Germination and Flowering Stages

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Laboratory of Breeding and Biotechnology, Institute of Plant Biology and Biotechnology, Timiryazev 45, Almaty 050040, Kazakhstan
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Faculty of Agrobiology, Kazakh National Agrarian Research University, Almaty 050010, Kazakhstan
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Tanir Research Laboratory, Almaty 050060, Kazakhstan
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A.I. Barayev Research and Production Center for Grain Farming, Shortandy 021600, Kazakhstan
Biology2026, 15(5), 384;https://doi.org/10.3390/biology15050384
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Simple Summary

This study evaluated the drought tolerance of doubled haploid (DH) lines derived from interspecific hybrids between rapeseed (Brassica napus) and turnip rape (Brassica rapa), comparing them to their parental lines. Under drought conditions simulated by PEG-6000 during seed germination and controlled irrigation at seedling and flowering, the DH lines showed better water retention and more robust antioxidant activity. Measurements of physiological parameters (CAT, POD, H2O2, MDA, Fv/Fm) along with analysis of drought-responsive gene expression confirmed the superior drought tolerance of these DH lines. The findings indicate that producing uniform DH lines from distant canola hybrids is an effective strategy to enhance drought tolerance, providing a valuable method for developing stress-tolerance crops.

Abstract

Drought stress is a major limiting factor for canola production in arid and semi-arid regions, particularly during seed germination, seedling and flowering stages. In this study, we evaluated drought responses of doubled haploid (DH) lines derived from interspecific hybrids of B. napus × B. rapa and their parental cultivars under simulated (PEG-6000) and soil-based drought conditions. Drought stress significantly reduced germination, growth, and physiological performance in all genotypes; however, DH lines consistently exhibited superior tolerance. Under PEG-induced osmotic stress, DH lines maintained higher germination rates, root elongation, and relative water content compared with parental genotypes. During seedling and flowering stages drought, DH lines showed lower accumulation of hydrogen peroxide and malondialdehyde, alongside markedly higher antioxidant enzyme activities (CAT and POD) and improved photosynthetic efficiency (Fv/Fm). Gene expression analysis revealed strong induction of drought-responsive genes, including WRKY28, MYB, LTP, WSP, metallothionein, and protein kinase family genes, particularly in DH lines at prolonged stress exposure. Multivariate analyses (PCA and correlation) confirmed a close association between enhanced antioxidant capacity, transcriptional activation, and drought tolerance traits. Overall, our results demonstrate that homozygous doubled haploid lines derived from distant hybridization between B. napus and B. rapa exhibit enhanced drought tolerance at both early and reproductive stages. These genotypes represent valuable genetic resources for breeding drought-tolerance canola cultivars.

1. Introduction

Oilseed crops play a crucial role in global food security due to their high nutritional and economic value. In Kazakhstan, oilseed cultivation has expanded in recent years; however, the availability of locally adapted rapeseed (B. napus L.) varieties remains limited, and commercial hybrids are unavailable [1]. Canola seeds (B. napus and B. rapa) are valuable for their low erucic acid and glucosinolate content, which makes their oil edible and sought after in the global market.
In the northern regions of Kazakhstan, where rapeseed is cultivated without irrigation, the impact of abiotic stresses, such as precipitation deficits and rising temperatures during the growing season, is intensifying. Under these conditions, turnip rape (B. rapa) is promising, as it has a short growing season and is resistant to abiotic stress factors in the environment; however, its yield is inferior to that of rapeseed in favorable years. One potential solution is to create interspecific hybrids and cultivars that combine the productivity of rapeseed with the resistance of turnip rape [2]. In contrast to Canada, Ukraine, and the European Union, the primary constraint on rapeseed cultivation in Kazakhstan is the arid growing conditions. Although drought can occur at any time during the growing season, two main periods of drought are more likely to occur: early drought, which coincides with seed germination and emergence, and drought during flowering, which affects seed setting and filling [2]. Seed germination is crucial for seedling formation, determining successful crop production [3] and, at the same time, being the most sensitive stage of the plant life cycle [4]. Crop formation depends on the interaction between the seedbed environment and seed quality [5,6]. In other words, any unfavorable conditions in the seedbed environment, such as drought stress, can compromise crop development [7] and subsequently seed yield [8]. Today, various methods are used for the breeding and production of new cultivars of canola (B. napus and B. rapa) with improved quantitative and qualitative agronomic characteristics, including increased resistance to abiotic stresses such as drought, resulting from the use of innovative biotechnological and genetic approaches [2,9,10].
Previous studies have shown genetic variability in the response of Brassica species to water stress during germination and early seedling growth stages [11,12,13]. However, evaluating drought tolerance in the field is often complicated by spatial and temporal variability in environmental factors [14,15,16]. As a result, controlled experimental approaches are widely used to simulate water stress during germination and early growth [17,18,19]. Polyethylene glycol (PEG-6000) is commonly used to induce osmotic stress in vitro, as it reduces water potential without penetrating plant tissue, thereby providing a stable and reproducible drought simulation. This method allows for the reliable assessment of genotypic responses to water stress at early stages of development [20,21,22].
Drought stress is often associated with the accumulation of reactive oxygen species (ROS), such as hydrogen peroxide (H2O2) and malondialdehyde (MDA). ROS cause serious damage to membrane properties and chlorophyll structure, which affects the normal metabolism of plant cells [23]. Plants possess an antioxidant defense system that eliminates ROS. Peroxidase (POD) protects the cell membrane from oxidative damage by removing excess ROS from cells under stress [24], whereas catalase (CAT) mitigates the destructive effects of stress [25].
Drought resistance is a multifaceted trait characterized by the intricate interplay of morphological, physiological, and molecular factors during periods of drought stress [26,27]. Genetic variability of Brassicaceae species during water deficit in critical phases of bud formation and flowering confirms the complexity of this trait and demonstrates the importance of studying morphophysiological characteristics during droughts [11,28,29]. Numerous plant genes and transcription factors associated with tolerance to drought and other abiotic stressors have been identified. Genes encoding late embryogenesis proteins have been shown to significantly enhance plant resistance to drought and salt stress [30]. Transcription factors, such as WRKY and bZIP, protein kinases like MAPK and CDPK, enzymes involved in abscisic acid biosynthesis (ABC) and gene families associated with the auxin signaling pathway (BnARP), play crucial roles in the study of gene expression during drought and other abiotic stresses [31,32]. Several studies have indicated that drought stress induces differentially expressed genes (DEGs), primarily including transcription factors (TFs), hormonal signaling pathways (e.g., ABA-dependent), stress protection proteins, detoxification-related genes, and photosynthesis-associated genes [33,34,35]. These DEGs play key roles in elucidating the molecular mechanisms underlying plant responses to drought, such as osmotic adjustment, reactive oxygen species (ROS) scavenging, and maintenance of photosynthetic efficiency. For instance, in a seminal study by Chen et al. [36], over 500 genes responsive to high-salinity and drought stress were identified from B. napus cDNA libraries using microarray hybridization. Subsequent validation via Northern blotting and quantitative RT-PCR confirmed differential expression profiles under abiotic stresses, highlighting candidate genes involved in stress signaling and tolerance in rapeseed.
The selection and breeding of cultivars with robust drought tolerance during the germination stage and early seedling growth will contribute to more stable canola production. Consequently, the objective of this study was to evaluate and compare the responses of rapeseed, turnip rape, and their interspecies hybrids to drought stress during germination, early seedling growth, bunting, and flowering under.

2. Materials and Methods

2.1. Plant Material

This study utilized six genotypes: two doubled haploid (DH) interspecific hybrid lines (B. napus × B. rapa)—DHKZ and DHGY, which were developed in our previous studies using haploid technology [19] and their four parental lines: B. napus cultivars: Galant and Kris, B. rapa cultivars: Zolotistaya and Yantarnaya. The main characteristics of these lines are summarized in Table 1.
Table 1. Main characteristics of homozygous lines studied.
Parental genotypes were used as controls and included two rapeseed cultivars, Kris and Galant (All-Russia Research Institute of Agricultural Biotechnology, Moscow, Russia), and two turnip rape cultivars, Zolotistaya and Yantarnaya (Federal State Budgetary Scientific Institution “V.S. Pustovoit All-Russian Research Institute of Oil Crops”, Krasnodar, Russia).

2.2. Seed Treatment with PEG-6000

Seeds were surface-sterilized in 0.1% NaOCl for 20 min and thoroughly rinsed with distilled water for 15 min. Drought stress was simulated using polyethylene glycol (PEG-6000). Three osmotic stress levels (8%, 10%, and 15% PEG) were applied according to Xie et al. [37], while distilled water served as the control.
The experiment followed a completely randomized two-factor factorial design with three biological replicates and 20 seeds per replicate.

2.3. Germination Test and Seedling Growth Parameters

Germination percentage (GP, %) was recorded at 3, 5, and 7 days after sowing. Germination was defined as the emergence of a sprout ≥ 3 mm in length. Shoot length (SL, cm) and root length (RL, cm) were measured on the eighth day after germination, marking the end of the experiment [35,38].

2.4. Relative Water Content (RWC)

RWC was determined using the third and fourth fully expanded leaves at the five-leaf stage. Fresh weight (FW) was recorded immediately after sampling. Leaves were immersed in distilled water for 12 h at room temperature to obtain turgid weight (TW), gently blotted dry, and weighed. Dry weight (DW) was obtained after drying samples at 70 °C for 48 h until constant mass [38,39].
RWC was calculated as:
RWC   ( % ) = FW − DW TW − DW × 100
Measurements were conducted with three biological replicates, each containing three leaves per plant.

2.5. Determination of Plant Drought Tolerance Under Controlled Soil Conditions

For soil-based drought treatment, pots were filled with equal amounts of soil and initially supplied with the same volume of water. Germinated seedlings were grown under control conditions until the five-true-leaf stage, after which irrigation was withheld for 14 days [40]. Plant survival was assessed three days after rewatering, and survival rates were calculated as the percentage of recovered plants. Measurements of antioxidant activity and gene expression were performed during two growth stages, namely seedling and flowering. At least three biological replicates were used per treatment.

2.5.1. Determination of Hydrogen Peroxide (H2O2) and Malondialdehyde (MDA) Contents

H2O2 content was determined following Batool et al. [41]. Leaf tissue (0.5 g) was homogenized in 5 mL of 0.1% trichloroacetic acid (TCA) and centrifuged at 12,000 rpm (16,128× g) for 20 min. An aliquot (0.5 mL) of the supernatant was mixed with 0.5 mL of 50 mM phosphate buffer (pH 7.0) and 1 mL of 1 M KI. Absorbance was measured at 390 nm, and H2O2 concentration was calculated using a standard curve.
MDA content was determined according to Liu et al. [42]. Leaf tissue (0.5 g) was homogenized in 5 mL of 0.1% TCA and mixed with 0.5% TBA in 20% TCA. The mixture was incubated at 95 °C for 30 min, cooled in an ice bath, and centrifuged at 11,200× g for 10 min. Absorbance was recorded at 532 and 600 nm, and MDA content was calculated using the extinction coefficient.

2.5.2. Measurement of Antioxidant Enzyme Activity and Photosynthetic Efficiency

Catalase (CAT, EC 1.11.1.6) activity was determined by monitoring H2O2 decomposition at 240 nm [43]. The reaction mixture (3 mL) contained 50 mM phosphate buffer (pH 7.0), 10 mM H2O2, and 100 µL enzyme extract. CAT activity was expressed as µmol H2O2 min−1 mg−1 protein using an extinction coefficient of 39.4 M−1·cm−1.
Peroxidase (POD, EC 1.11.1.7) activity was measured using a modified guaiacol method. The 3 mL reaction mixture contained 50 mM phosphate buffer (pH 6.0), 20 mM guaiacol, 10 mM H2O2, and 100 µL enzyme extract. POD activity was expressed as ΔA470 min−1 mg−1 protein. All enzymatic assays were performed in triplicate.
Photosynthetic efficiency (Fv/Fm) was measured using a pulse-amplitude modulation fluorometer (IMAG-MAXI, Heinz Walz, Effeltrich, Germany). Four biological replicates, each with three technical measurements, were used.

2.5.3. Analysis of Candidate Gene Expression

Eight candidate genes related to abiotic stress responses were selected [44,45,46] (Table 2). Total RNA was extracted from 0.1 g leaf tissue using TRI Reagent (Molecular Research Center, Cincinnati, OH, USA), and cDNA synthesis was performed using the Fast Quant RT Kit (TIANGEN, Beijing, China). Primers were designed using Primer 5.0 and Primer 3Plus (18–22 nt, Tm 58–62 °C, amplicon 100–200 bp) (Table 2).
Table 2. Primer sequences for RT-PCR.
qRT-PCR was performed on a CFX96 system (Bio-Rad, Hercules, CA, USA) with SYBR Green (SuperReal PreMix, TIANGEN). Amplification: 95 °C for 15 min, followed by 40 cycles of 95 °C for 10 s and 60 °C for 32 s. BnACT2 was used as a reference gene after validation for stable expression. Primer efficiency (95–105%) and melting curves were checked to ensure specificity. Each sample was analyzed with three technical replicates per three biological replicates. Relative expression was calculated using the 2−ΔΔCT method [39].

2.6. Statistical Analysis

ANOVA was used to analyze data. Significant differences were further evaluated by Duncan’s multiple range test (DMRT). Pearson’s correlation assessed relationships among parameters. PCA was performed on standardized data (Z-score) using RStudio v.2026.01.1+403 with FactoMineR v.2.12 and factoextra packages v.1.0.7. All analyses were cross-checked in SPSS v.23.

3. Results

3.1. Effects of Osmotic Stress on Germination and the Seedling Stage

Variance analysis (ANOVA) revealed that both osmotic stress (PEG-induced drought) and genotype had significant effects on seed germination, shoot length, root length, and relative water content (RWC) in leaves (Table 3). The interaction between genotype and stress was also significant, indicating that different genotypes respond variably to water deficit. Overall, drought had a stronger effect than genotype on these traits, particularly RWC and shoot growth, highlighting the sensitivity of these parameters to water stress.
Table 3. ANOVA for seed germination and seedling-related traits and relative water content of rape seed genotypes evaluated under drought conditions.
This indicates that parental cultivars and DH lines respond differently to drought conditions, and these differences are genetic.
Figure 1A shows that for all genotypes, a decrease in water potential resulted in a significant decrease in germination. The germination rate in the control (0% PEG) reached 100% for all genotypes, whereas under stress conditions, the minimum values (32.0, 63.3, 33.3, 61.7, 69.0, and 86.7%) were observed at high PEG concentrations (15% PEG), and the maximum values (66.3, 78.3, 64.0, 78.3, 88.3, and 96.7%) were observed under moderate stress (8% PEG).
Figure 1. Effect of polyethylene glycol (PEG) on seed germination and morphophysiological parameters in parental cultivars and (doubled-haploid) DH lines: (A) Seed appearance at different PEG concentrations (0–15%); (B) Germination; (C) Root length (RL); (D) Relative water content (RWC); (E) Shoot length (SL). The data shown are the means of three replicates (SD) based on Duncan’s multiple range test.
Under drought conditions, germination and seedling growth declined in all genotypes, but the doubled-haploid lines DHKZ and DHGY maintained higher germination rates and better growth compared to their parental cultivars. Among them, DHGY exhibited the highest germination across all stress levels, demonstrating superior osmotic stress tolerance. Both DH lines also maintained higher RWC under increasing PEG concentrations, whereas parental genotypes showed notable declines in leaf hydration. Root elongation and shoot growth were better preserved in DH lines under moderate and high drought, confirming their greater resilience during early seedling development.
Under drought conditions, the overall mean relative water content (RWC) in leaves gradually decreased as stress levels increased (8, 10, and 15% PEG) (Figure 1D). The water content in the leaves was high in control (0% PEG) and 89.3, 93.7, 94.0, 91.3, 93.7, and 90.3%, in the genotypes Kris, Zolotistaya, Galant, Yantarnaya, DHKZ, and DHGY, respectively. Under drought conditions, the highest RWC was found in the DHKZ line (90.5%, respectively) under 8% PEG stress, as well as in plants of the Zolotistaya (87%) and Yantarnaya (80.7%) turnip rape cultivars at the same stress level. However, with an increase in osmotic potential (10 and 15% PEG), the RWC values of rapeseed and turnip rape plants decreased significantly, while the DHGY line showed stability at all stress levels (72.3% at 8% PEG, 72.7% at 10% PEG, and 73.3% at 15% PEG, respectively). It should be noted that in plants of the DHKZ line, the RWC value decreased depending on the PEG concentration (79% at 10% PEG and 74.3% at 15% PEG, respectively) but exceeded that of the parent genotypes.
In the absence of stress, the Kris rapeseed cultivar had the highest average SL values (4.3 cm), followed by the Zolotistaya turnip rape cultivar and the Galant rapeseed cultivar (4.2 and 4.0 cm, respectively), while the SL values for the DHKZ and DHGY lines ranged from 3.7 to 4.1 (Figure 1C). At an osmotic potential of 8–10% PEG, a sharp decrease in SL was observed in all genotypes studied, while DH lines were able to develop shoots at PEG-induced osmotic potentials of 10 and 15%. Similarly, PEG-induced stress led to a significant decrease in RL in the original genotypes compared to the studied lines (Figure 1E). With increasing drought levels, root elongation was observed in DH lines. A significant increase in RL was observed at 10% PEG in the lines, 19.4 cm in DHKZ and 20.2 cm in DHGY, respectively. The root length of rapeseed cultivars at an osmotic potential of 10% PEG was approximately 6 cm, and at 15% PEG, it ranged from 4.8 to 5.3 cm for Kris and Galant. While turnip cultivars showed average RL values, they ranged from 9 to 10 cm for both cultivars at 10% PEG, and at 15% drought, they ranged from 5.7 (Zolotistaya) to 6.8 cm (Yantarnaya).

Correlations of Traits Under Control and PEG-6000 Stress

Based on data obtained under osmotic stress (PEG) conditions, PCA1 and PCA2 components explained 68.6% and 17.1% of the total variance, respectively, which together account for 85.7%—a high value (Figure 2A). The DHGY and DHKZ lines were clearly grouped in the upper right quadrant and showed a high association with traits such as RL, SL, RWC, and germination. These parameters suggest good water status and plant growth, which may indicate a high degree of DH line resistance to PEG-induced drought. At the same time, the Galant, Kris, and Yantarnaya genotypes were located predominantly in the lower left quadrant, demonstrating opposite characteristics, suggesting lower resistance to osmotic stress. The Zolotistaya genotype occupied an intermediate position between the other groups.
Figure 2. Multivariate analysis of morphophysiological traits: (A) PCA based on germination, root length (RL), shoot length (SL), and relative water content (RWC). Doubled-haploid (DH) lines (DHKZ, DHGY) were grouped separately, demonstrating higher stress adaptation. (B) Correlation matrix between the traits studied.
The heat map in Figure 2B demonstrates the relationship between the main morphophysiological characteristics of seed germination of the studied objects during drought. Principal component analysis (PCA) separated DH lines from parental genotypes, with DHGY and DHKZ clustering in association with traits reflecting high water status, growth, and drought resistance. In contrast, parental cultivars clustered separately, reflecting lower tolerance. Correlation analysis showed strong positive relationships among germination, RWC, and seedling growth (r = 0.4–0.8), confirming the central role of tissue water status in early drought response.
These results indicate that DH lines better maintain hydration in seedlings under simulated drought conditions, demonstrating an increased ability to conserve water compared to the original rapeseed and turnip rape genotypes. Thus, the DHGY and DHKZ lines show the strongest positive correlation with key traits of PEG stress resistance and are potentially tolerant to osmotic stress.

3.2. Activities of Enzymatic Antioxidants, Variations in Photosynthetic Activities, and MDA and H2O2 Contents Under Drought Stress

To further study the response of plants in the seedling and flowering phases under drought conditions, they were subjected to drought stress under controlled conditions by limiting irrigation for 14 days. Visual evaluation of rapeseed genotypes at the seedling stage showed varying responses to water stress (Figure 3). In the control plants, all samples retained normal turgor and healthy leaf surface, while under drought conditions, the Galant and Kris cultivars showed severe wilting and leaf curling (Figure 3). The Zolotistaya and Yantarnaya genotypes were characterized by moderate sensitivity, manifested in partial wilting and yellowing of the leaves. DHGY and DHKZ proved to be more resistant, with less pronounced wilting and curling of leaves, and with most of the leaf biomass remaining green.
Figure 3. Phenotypic manifestations of the parental cultivars and DH (doubled-haploid) lines under irrigation (control) and non-irrigation (drought) conditions.
Physiological and biochemical analyses showed that drought causes a significant increase in the content and the activity of antioxidant enzymes such as POD and CAT, as well as H2O2, MDA, and photosynthetic activity (Fv/Fm), which was analyzed in all genotypes studied and showed a significant difference in their response to stress (Table 4). It was found that the content of H2O2 and MDA significantly increased (p < 0.0001) in all variants, especially in the initial genotypes—the rapeseed and turnip rape cultivars. The highest values of these indicators were recorded at 72 h of drought (Figure 4). An increase in H2O2 and MDA was also observed in DH lines, but their levels were 0.5–1.2 times lower than in the parental cultivars.
Table 4. ANOVA for oxidative stress and physiological parameters of rapeseed genotypes evaluated under drought conditions.
Figure 4. Impact of drought on hydrogen peroxide (H2O2), malondialdehyde (MDA) content, peroxidase (POD), catalase (CAT), and photosynthetic II efficiency (Fv/Fm) in parental cultivars and DH (doubled haploid) lines. According to Duncan’s test, different letters above the bars indicate significant differences (p < 0.001).
Enzyme activity measurements showed that the activity levels of important antioxidants increased sharply in DH lines compared to the original genotypes at 24 h of drought (Figure 4). Physiological and biochemical assessments at the flowering stage showed that drought increased oxidative stress markers (H2O2, MDA) and antioxidant enzyme activities (CAT, POD) in all genotypes. DH lines displayed higher antioxidant activity and lower accumulation of H2O2 and MDA compared to parental plants, demonstrating better protection against oxidative damage. Photosynthetic efficiency (Fv/Fm) decreased under drought, but DH lines retained 2–3 times higher activity than parental genotypes. For POD, a gradual increase in activity was observed every 24 h, reaching 250% in the DHKZ line and 270% in the DHGY line on the third day of drought, while in rapeseed cultivars, this indicator was within 105%, and in turnip cultivars, they were 109% for Zolotistaya and 140% for Yantaraya, respectively.
According to our data (Table 3), drought stress significantly (p < 0.0001) reduced Fv/Fm in all genotypes studied. However, Fv/Fm activity in DH lines decreased from 93 to 60% in DHGY and from 86 to 50% in DHKZ, while in parental genotypes, it decreased to 13% in rapeseed plants and to 20% and 27% in the plants of the turnip rape cultivars Yantarnaya and Zolotistaya, respectively. As a result, photosynthetic activity in DH lines was 2–3 times higher than in parental forms.

3.3. Expression Analysis of Stress-Induced Genes

The study found that the expression level of the genes studied varied significantly depending on the time of treatment and genotype (parents of the cultivar and DH lines). Figure 5 illustrates the change in expression of eight genes associated with stress response in parental genotypes and lines at different times of drought exposure (0, 24, 48, and 72 h). Gene expression analysis revealed that stress-responsive genes, including WRKY, MYB, metallothionein, LTP, and others, were strongly induced in DH lines during drought, particularly at 48–72 h, while parental cultivars showed weaker or irregular activation. This enhanced transcriptional response in DH lines correlates with their physiological resilience and improved water status.
Figure 5. Relative expression levels of Lipid transfer protein (LTP), WRKY, Metallothionein (MT), MYB-like transcription factor, Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), Protein kinase, ARP gene, and WSP gene in parental cultivars and doubled haploid (DH) lines under drought. According to Duncan’s test, different letters above the bars indicate significant differences (p < 0.001).
Active regulation of the protein kinase, ARP, and WSP genes was also observed, especially in DHKZ and DHGY, which may indicate their involvement in signaling cascades and adaptive mechanisms during water deficiency. While in certain cultivars (especially Kris and Galant), the changes in expression were minimal and irregular, in resistant DH lines, a clear and time-dependent pattern of activation was observed. These data confirm the key role of these genes in the formation of drought resistance and highlight the potential of DHKZ and DHGY as genetic resources for further breeding.

3.4. Principal Component Analysis (PCA) and Correlation Analysis in B. napus Cultivars Under Drought Stress

Principal component analysis (PCA) was used to study the relationship between traits and identify stable, promising genotypes based on multiple characteristics. Under drought stress conditions, the first principal component, PCA 1, explains about 63.2% of the variation, while the second principal component, PCA 2, accounts for 23.2% (Figure 6A). Two clusters are clearly distinguished on the biplot: the DHGY and DHKZ lines are located in the positive part of Dim1 and are associated with increased values of H2O2 and MDA, which reflects increased oxidative stress, and also correlate with the activity of the antioxidant enzymes CAT and POD and the transcription factor WRKY, indicating the activation of defense mechanisms. At the same time, the Galant, Kris, Yantarnaya, and Zolotistaya genotypes are present in the negative part of Dim1 and are associated with Fv/Fm indicators, demonstrating higher biomass and less severe oxidative stress.
Figure 6. Multivariate and correlation analysis of physiological and gene expression traits under drought stress: (A) PCA plot based on physiological indicators (catalase (CAT), peroxidase (POD), hydrogen peroxide (H2O2), malondialdehyde (MDA), photosynthetic II efficiency (Fv/Fm) and the expression of stress-associated genes (LTP, WRKY, MYB, metallothionein, protein kinase, etc.). Doubled-haploid (DH) lines (DHKZ, DHGY) clustered separately from rapeseed and turnip rape cultivars, reflecting distinct stress-response profiles. (B) Correlation matrix of physiological and molecular traits. Strong positive correlations were observed among antioxidant enzyme activity and stress-related gene expression, while H2O2 and MDA were negatively associated with most adaptive traits.
The heat map in the figure illustrates the correlational relationships between antioxidant enzymes, oxidative stress markers, and the expression of stress-associated genes in parental genotypes and lines (Figure 6B). Negative correlations are observed between H2O2, MDA, and fresh weight (Fv/Fm) content, as well as with most of the genes studied, confirming their role as stress markers. At the same time, the expression of ARP, WSP, Protein kinase, Metalotheanin, LTP, MLTF, and WRKY genes positively correlates with CAT and POD activity, indicating their involvement in the activation of antioxidant defense. A particularly strong positive correlation can be seen between WRKY, LTP, and antioxidant enzymes, which emphasizes the contribution of these transcription factors to drought resistance. Thus, the heat map confirms the previously obtained results on gene expression and physiological responses of genotypes under water deficit.
Together, these results indicate that DHGY and DHKZ lines combine superior germination, seedling growth, water retention, antioxidant capacity, photosynthetic efficiency, and stress gene activation, making them promising genotypes for drought tolerance. Correlation and PCA analyses confirmed the coordinated regulation of physiological and molecular traits underlying their adaptation, while parental cultivars exhibited lower stress resilience and limited activation of protective mechanisms.

4. Discussion

In this study, drought stress simulated by PEG-6000 and soil water deficit significantly affected all analyzed morphophysiological, biochemical, and molecular parameters in both doubled haploid (DH) lines and parental genotypes. However, the magnitude and nature of these responses differed markedly between genotypes, indicating substantial genetic variation in drought tolerance mechanisms.

4.1. Drought Effects on Germination and Early Seedling Development

PEG-induced osmotic stress caused a pronounced reduction in seed germination, shoot length (SL), root length (RL), and relative water content (RWC) in all genotypes, with parental cultivars being more strongly affected than DH lines (Figure 1). These results are consistent with previous studies demonstrating that drought stress restricts water uptake, limits metabolic activity, and suppresses early seedling growth in Brassica species [12,13,35]. At moderate osmotic stress (8% PEG), all genotypes maintained relatively high germination; however, at severe stress levels (15% PEG), only DH lines retained high germination capacity, while parental rapeseed cultivars were strongly inhibited. Similar stress-dependent reductions in germination have been reported in mustard [47], sunflower [48], sesame [49], and safflower [50].
The ability of DHKZ and DHGY lines to maintain higher germination rates under severe osmotic stress suggests improved seed vigor and water uptake efficiency. Drought stress typically disrupts enzymatic activity and energy metabolism during germination, resulting in reduced seedling establishment [50,51]. In the present study, DH lines demonstrated enhanced root elongation under increasing drought severity, reaching up to 20.4 cm at 10–15% PEG, whereas parental genotypes exhibited significantly shorter roots. Similar drought-induced root elongation has been reported in rapeseed and is considered an adaptive strategy enabling access to deeper soil moisture [52].
Notably, only DH lines were capable of sustained shoot development under moderate and severe PEG stress, indicating superior allocation of resources to maintain growth. The increase in root-to-shoot ratio observed across all genotypes under drought is consistent with previous reports and likely reflects preferential carbon and nutrient allocation toward roots at the expense of shoot growth under water deficit conditions [53]. Overall, these findings indicate that DH lines possess enhanced early-stage drought adaptation mechanisms compared with parental rapeseed and turnip rape cultivars.

4.2. Oxidative Stress and Antioxidant Defense Under Drought

Drought stress is closely associated with excessive accumulation of reactive oxygen species (ROS), leading to oxidative damage of cellular membranes and metabolic dysfunction. In the present study, drought significantly increased hydrogen peroxide (H2O2) and malondialdehyde (MDA) contents in all genotypes, with the highest levels observed in parental cultivars. Elevated H2O2 and MDA are widely recognized as indicators of oxidative stress and lipid peroxidation under water deficit conditions [54,55,56].
In contrast, DHKZ and DHGY lines accumulated significantly lower levels of H2O2 and MDA, indicating more efficient control of oxidative damage. This reduced ROS accumulation was accompanied by a pronounced activation of antioxidant enzymes Catalase (CAT) activity increased by 60.4–93.3% in DHKZ and 47.7–110.0% in DHGY, while peroxidase (POD) activity reached 250–270% by the third day of drought exposure. In parental genotypes, the increase in antioxidant activity was comparatively modest. These results agree with earlier studies showing that drought-tolerant rapeseed genotypes exhibit stronger antioxidant responses than sensitive ones [57,58,59].
Antioxidant enzymes such as CAT, POD, SOD, APX, and GPX play a central role in maintaining redox homeostasis and protecting cellular structures from oxidative injury during stress [60,61]. The enhanced enzymatic activity observed in DH lines likely contributes to their improved drought tolerance by limiting ROS-induced cellular damage.

4.3. Photosynthetic Performance Under Water Deficit

Photosynthetic efficiency, assessed via the maximum quantum yield of photosystem II (Fv/Fm), declined significantly under drought stress in all genotypes. However, DH lines maintained substantially higher Fv/Fm values than parental rapeseed and turnip rape cultivars. Reductions in Fv/Fm are commonly associated with impaired PSII photochemistry, stomatal closure, and restricted CO2 diffusion under drought stress [62,63,64,65].
The comparatively higher Fv/Fm values in DH lines suggest better preservation of photosynthetic apparatus integrity and more effective stress acclimation. Previous studies have demonstrated that sustained photosynthetic activity under drought contributes to improved biomass accumulation and yield stability in crops such as rice and wheat [66,67]. In rapeseed, high photosynthetic efficiency is positively associated with seed yield and oil content, particularly under suboptimal environmental conditions [68]. Therefore, the ability of DH lines to maintain higher photosynthetic performance under drought represents a key adaptive advantage.

4.4. Transcriptional Regulation of Drought Responses

To elucidate molecular mechanisms underlying the observed physiological differences, the expression of eight drought-responsive genes was analyzed. The DHKZ and DHGY lines exhibited strong and time-dependent upregulation of key regulatory and protective genes, including WRKY28, MYB, LTP, WSP, metallothionein, protein kinase family genes, and GAPDH. In contrast, parental cultivars showed weaker and less consistent transcriptional responses.
Notably, GAPDH family members in plants, including B. napus, exhibit differential expressions under various abiotic stresses such as drought, salinity, and oxidative stress. Beyond their classical role in glycolysis, these proteins contribute to redox homeostasis, signaling, and metabolic adjustments [69,70]. Recent studies in B. napus have identified GAPDH genes containing promoter elements responsive to abiotic stresses (including drought), with their expression modulated by phytohormones and exhibiting crosstalk with drought-related pathways [69]. In other crops, specific GAPDH isoforms are induced by drought and positively enhance tolerance upon overexpression, for instance, Gh_GAPDH9 in cotton and TaGAPC1 in wheat (regulated by WRKY40), which improve plant survival, ROS management, and osmotic adjustment [71,72]. This multifunctional (moonlighting) role of GAPDH—encompassing interactions with transcription factors, ROS scavenging, and stress signaling—explains its inclusion here as a drought-responsive gene, alongside canonical regulatory transcription factors (WRKY, MYB) and protective proteins (metallothionein, LTP, WSP).
WRKY transcription factors are well-established regulators of plant responses to abiotic stress, including drought, salinity, and temperature extremes [73,74,75,76]. The pronounced induction of WRKY genes in DH lines is consistent with their proposed role in coordinating antioxidant defense and stress signaling pathways [75,76]. Similarly, MYB transcription factors are known to regulate ABA-dependent signaling and drought-responsive gene networks in Brassica species and Arabidopsis [77,78].
Metallothioneins function as metal-binding antioxidants and play an important role in ROS scavenging under stress conditions. Their positive association with drought tolerance has been reported in rapeseed [79], oats [80], chickpea [81], and sweet potato [82], in agreement with the present findings. Elevated expression of protein kinase genes in DH lines further suggests enhanced activation of ABA-mediated signaling cascades, consistent with previous reports describing the role of CPK and MAPK pathways in drought adaptation [83].
Notably, many of these genes are considered conserved components of drought response networks across plant species, including soybean, rice, and maize [83,84,85], highlighting the broader relevance of the transcriptional patterns observed in DH lines.

4.5. Multivariate Analysis and Integrative Drought Tolerance Mechanisms

Principal component analysis (PCA) and correlation analysis provided an integrative view of genotype performance under drought stress. DHKZ and DHGY clustered distinctly from parental genotypes and were strongly associated with favorable traits such as RL, SL, germination, RWC, antioxidant enzyme activity, and drought-responsive gene expression. Similar clustering patterns have been reported for drought-tolerant rapeseed and other crop species under PEG-induced stress [86,87,88].
The strong positive correlations between antioxidant enzyme activity (CAT, POD) and transcription factors (WRKY, MYB, LTP) emphasize the coordinated regulation of physiological and molecular responses in DH lines. Combining osmotic stress assays with soil-based drought experiments, as performed in this study, enhances the robustness of drought tolerance assessment and has been recommended for reliable genotype screening [89]. Nevertheless, further validation under field conditions is required to confirm the performance of these lines in complex and variable environments.
The superior drought tolerance of the DHGY and DHKZ lines compared to their parental cultivars may be genetically attributed to several mechanisms inherent to interspecific hybridization and doubled haploid production. First, the DH process rapidly fixes homozygous combinations of alleles through chromosome doubling from haploid microspores, allowing the capture and stabilization of rare, favorable recombinant genotypes that are heterozygous or infrequent in the F1 hybrid [90]. Second, interspecific crosses between B. napus (AACC) and B. rapa (AA) enable introgression of adaptive genomic segments from the A genome of B. rapa, which often exhibits stronger drought responses during germination and early growth than B. napus (e.g., higher germination rates and RWC under severe PEG in Yantarnaya and Zolotistaya parents; see also recent advances in Brassica polyploidy and introgression [91,92]). Such introgressions may enhance osmotic adjustment, root plasticity, or ROS scavenging capacity observed in our DH lines, as supported by studies on climate resilience via wild relatives and interspecific gene flow in Brassica [93]. Third, homoeologous exchange and recombination during DH production can generate novel allelic combinations, fixing transgressive segregation for drought-related traits and reducing genetic load through elimination of deleterious alleles [93]. These genetic factors likely underline the coordinated physiological (enhanced antioxidant defense, photosynthetic maintenance) and transcriptional (stronger upregulation of WRKY, MYB, metallothionein) advantages documented here, highlighting the value of interspecific DH lines for breeding drought-resilient rapeseed.
Consequently, a thorough investigation revealed that the DHGY and DHKZ lines exhibit strong drought resistance, which is supported by the antioxidant system’s active operation in situations of elevated oxidative stress. Additionally, Galant and Kris have demonstrated a high level of drought sensitivity. The turnip rape cultivars Yantarnaya and Zolotistaya showed intermittent stability, with indicators that were comparatively stable for the parameters under study and less obvious stress responses. While this study provides robust evidence of drought tolerance in controlled conditions, limitations include the reliance on lab-based simulations (e.g., PEG-6000), which may not fully replicate field variability such as soil heterogeneity or multi-stressor interactions. Additionally, the analysis is limited to a small number of genotypes (two DH lines and four parents), potentially overlooking broader genetic diversity. Future work could address these by conducting multi-location field trials and expanding to whole-genome sequencing for marker-assisted selection.

5. Conclusions

Collectively, the results demonstrate that DHKZ and DHGY doubled haploid lines exhibit enhanced drought tolerance through coordinated regulation of plant water status, antioxidant defense, photosynthetic performance, and stress-responsive gene expression. In contrast, rapeseed cultivars Galant and Kris were highly drought-sensitive, while turnip rape cultivars Yantarnaya and Zolotistaya showed intermediate and less stable responses to drought stress.
These findings highlight the strong potential of interspecific doubled haploid lines as valuable genetic resources for breeding drought-tolerance canola. Although the present study provides robust evidence under controlled conditions, further validation through multi-location field trials and expanded genetic analyses will be essential to fully exploit these genotypes in practical breeding programs.

Author Contributions

Conceptualization, A.D.; methodology, A.D., Z.A., I.O. and E.T.; software, D.D. and R.K.; validation, A.D. and M.T.; formal analysis, A.D. and D.D.; investigation, M.S. and Z.S.; writing—original draft preparation, A.D. and K.Z.; writing—review and editing, A.D. and K.Z.; supervision, A.D., K.Z., M.S. and Z.S.; project administration, M.S. and Z.S.; funding acquisition, K.Z., M.S. and Z.S. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the Committee of Science of the Ministry of Science and Higher Education of the Republic of Kazakhstan (grant no. АР26102771).

Institutional Review Board Statement

Not applicable.

Data Availability Statement

The original contributions presented in this study are included in the article. Further inquiries can be directed to the corresponding author.

Conflicts of Interest

The authors declare no conflicts of interest.

Abbreviations

The following abbreviations are used in this manuscript:
DHDoubled haploid
RLRoot length
SLShoot length
RWCRelative water content
GRGermination rate
H2O2Hydrogen peroxide
MDAMalondialdehyde
PODPeroxidase
CATCatalase
FV/FMPhotosynthetic II efficiency

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