Thermal Stress as a Tool for Manipulating Haploid Induction Rate in Brassica napus

Summary statement

This study reveals that short‐term low‐temperature stress improves the haploid induction rate (HIR) in rapeseed (Brassica napus). While high temperature produces normal‐sized and aborted seeds, low temperature specifically yields small‐sized seeds associated with elevated HIR. These findings demonstrate that environmental manipulation can substantially enhance HIR and offer a promising strategy for future haploid breeding research.

Rapeseed (Brassica napus) is a common oilseed crop in China, and China accounts for 20% of global rapeseed production, yet it is not self‐sufficient and imports about 60% of its rapeseed. To meet population demand on limited agricultural land, high‐yielding varieties are the primary objective. Haploid induction (HI) is a strong candidate technique for producing homozygous lines within a single breeding cycle. HI is broadly divided into two main categories: In vitro HI (including ovule, anther and microspore culture) and in vivo HI (including distinct hybridization and haploid induction lines (HIL)). Among these HI categories, only HIL is the most widely adopted method due to its broad applicability across crop species and operational simplicity (Quiroz et al. 2024). HIL have been developed in many crop plants by mutating different kind of key genes like genes involved in reproductive system of plants, that is, KINETOCHORE NULL2 Protein (KNL2), CENTROMERIC HISTONE H3 (CENH3), GAMETE EXPRESSION PROTEIN 1 (GEX1), EGG CELL SPECIFIC (ECS), GIGAS CELL1 (GIG1) and other key genes including MATRILINEAL/PHOSPHOLIPASE A1/NOT LIKE DAD (MTL/PLA1/NLD), POLLEN SPECIFIC PEROXIDASE (POD65) and DOMAIN OF UNKNOWN FUNCTION 679 MEMBRANE PROTEIN (DMP) (Quiroz et al. 2024; Bakhsh et al. 2025, 2026). Although HIL plays a significant role in haploid breeding, without hindrance to genotypic specificity and technical labour, it is still not commercialized on a large scale due to a low haploid induction rate (HIR), a major concern for most commercial companies. Hence, after extensive research on HIL, the primary objective is to increase HIRs in crop plants. The environment plays a key role in HIR, like temperature manipulation. CENH3‐based HI system in Arabidopsis, ECS‐based HI system in Arabidopsis, POD65‐based HI system in maize, and parthenogenesis‐based HI in sunflower have suggested that temperature plays a significant role in HI efficiency (Ahmadli et al. 2023; Wang et al. 2023; Yang et al. 2025; Zhao et al. 2025; Jiang et al. 2022; Lv et al. 2025). Our previous study reported that a mutation in EGG CELL SPECIFIC ENDOPEPTIDASES (ECS) in B. napus (rapeseed) leads to the development of a maternal haploid due to the unsuccessful fusion of parent's gamete cells (Bakhsh et al. 2026). According to Zhao et al. (2025), temperature plays a significant role in controlling HIR in the ECS‐based HI system. In our present study, we aim to clarify the ECS1‐HI system in rapeseed under temperature‐mediated conditions. Moreover, our aim is to assess the fertility of Bnaecs1 mutants under different temperature conditions, which are known to improve HIR. The effect of temperature was assessed on BnaECS1‐HI by transferring Bnaecs1 mutants (Figure S1A,B; Bakhsh et al. 2026) to different temperature environments, that is, low (16°C), normal (22°C) and high (30°C), after bolting for 2 weeks. Hybridization was performed using pollen from two WT varieties, that is, W2 and NO2127 (both are white‐flowered varieties). F1 seeds were screened for haploid identification through SSR marker‐based genotyping (Figure S2A), followed by ploidy analysis by using flow cytometry (Figure S2B,C) and finally whole genome resequencing by using a 50K SNP array chip (Figure S2D). All haploid plants exhibit a similar phenotype to the already reported phenotype of a haploid plant (Quiroz et al. 2024), that is, small and male sterile flowers (Figure 1A–E). Under normal temperature conditions (22°C), Bnaecs1 mutants induce 0.98%–1.09% HIR, consistent with our previous research (Bakhsh et al. 2026). At high temperature (30°C), we did not obtain any haploid plants in any cross combination of Bnaecs1 mutants with WT. Moreover, at low temperature (16°C), the HIR was increased, that is, ranged from 2.11% to 2.33% (more than doubled compared to standard conditions). These results confirmed that HIR was significantly affected by changes in temperature: high temperature reduced HIR, while low temperature increased HIR compared to normal conditions (Figure 1J).

Figure 1.

Haploid induction and seed setting at different temperatures module through BnaECS1 mutants. (A–E) Presenting diploid vs haploid flowers, floral parts and flower bunch, respectively, haploid plants exhibit male sterility, while diploid plants were normal, presented flowers belong to the F1 progeny of Bnaecs1 × NO2127, where NO2127 is a white flower colour variety, which is dominant over the yellow colour variety (inducer line). (F) Statistics of crossed seeds between Bnaecs1 mutant and WT varieties, where aborted seeds were maximum at 30°C and small seeds were maximum at 16°C. (G and H) Siliques of crossed seeds in different temperature stress condition after 14–20 days of pollination, where ‘*’ sign indicates the undeveloped or aborted seeds, while arrow head indicates the small‐sized seeds. (I) Type of seed classes observed during seed setting (normal seed, small seed and aborted seeds). (J) Haploid induction rate (HIR) under different temperature conditions, where Bnaecs1 was used as the female parent, while W2 and NO2127 were used as the male parent. All data were analyzed using a two‐tailed Student's t‐test. (ns, not significant, p > 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001; scale bar = 1 cm.

Our study further explores the mechanism underlying HIR manipulation by temperature, examining the seed set phenotype in Bnaecs1 mutants. Previous studies on the ECS‐HI system indicated that haploids resulted from failure of egg‐sperm fusion, leading to smaller or undeveloped seeds (Zhang et al. 2023). To conclude the HIR response to temperature manipulation, we have observed the phenotype of seeds under different temperature conditions. First, all F1 seeds were divided into three categories, that is, normal, small and aborted seeds, based on visual appearance. Next, we have measured the 1000 seeds weight and seed size of all categories and our results revealed that 1000 seeds weight of all categories were highly significantly different, that is, on average the normal category was 4.45 g, small category was 1.38 g and aborted category was 0.06 g (Figure S3A). Similarly, the seed size was also significantly different of normal was 0.0297 cm², small was 0.015 cm² and aborted was 0.0055 cm² on average (Figure S3B). At low temperature stress conditions (16°C), small‐sized seeds were increased up to 5 times as compared to plants grown at high temperature stress conditions (30°C) and 3 times as compared to normal temperature conditions (22°C), which may indicate that the fusion of both gametes was unsuccessful at low temperature. Moreover, undeveloped seeds/aborted seeds under low‐temperature conditions were similar to those under normal conditions, but were much lower than under high‐temperature stress conditions. Under high‐temperature (30°C) stress, aborted seeds were about 16% (Figure 1F–I). At high temperature (30°C), the number of small seeds decreased, while undeveloped and aborted seeds increased, indicating that high‐temperature stress drastically affects seed development. On the other hand, seed size was more affected by low‐temperature stress (smaller seeds were more numerous), whereas high‐temperature stress increased seed size. These results suggested that the reduction in smaller seeds under high‐temperature stress may be restore the failure of gamete cell fusion, along with a negative impact on seed growth, thereby affecting HI frequency (Figure 1J). Our results reveal that environmental factors, especially temperature, play a significant role in the fertilization process. Moreover, our study practically optimized the way in which HI can be increased/improved by utilizing different types of environments. Recently, multiple HI system describe the importance of temperature in HI, that is, facultative parthenogenesis in sunflower and ECS‐HI system in Arabidopsis both promote HIR with low temperature stress (Lv et al. 2025; Zhao et al. 2025), similar as our study observed in BnaECS1 HI system in B. napus but still the genetic basis of this low temperature based mediated HI is under question. On the other hand, CENH3‐based HI in Arabidopsis is induced by high‐temperature stress (Ahmadli et al. 2023; Wang et al. 2023; Yang et al. 2025). These types of responses, that is, low temperature mediate HIR or high temperature mediate HIR, suggest that the HI system or HIR are dependent on the type of mechanism that causes HI and the genetic background. Now it's important to understand how temperature affects the mechanism of HI, either gamete fusion, chromosomal defects or other possible gametophytic events. Although sperm–egg fusion failure provides a baseline for HI in Arabidopsis, including the ECS‐HI system and GEX1‐HI system (Zhang et al. 2023; Sun et al. 2025). Therefore, the mystery of gamete cell fusion's temperature dependence will offer a novel topic for improving HIR in different crops. Many factors involved in gamete cell fusion, that is, GEX1, actin, GCS1, and heat shock proteins, are thermally sensitive (Zhao et al. 2025). Moreover, the endoplasmic reticulum (ER) system, including BiP1 (immunoglobulin‐binding proteins), HS protein and several cofactors, including ERdj3A and ERdj3B, has been important for karyogamy and nuclear fusion (Maruyama et al. 2019). All the discussed findings and our results not only offer a potential guideline for improving HI systems in crop plants but also highlight the role of temperature in gamete fusion which will provide a new way for future research.

In conclusion, our results revealed that the BnaECS1‐HI system in rapeseed is mediated by temperature fluctuation. HIR is increased by low‐temperature mediation, more likely due to the production of small seeds, and possibly resulting from gamete fusion failure, which are decreased/inhibited by high temperature due to its negative effect on seed development/plant fertility. For efficient commercial use of the HI system across a broad range of crops, it is important to understand how environmental factors influence HIR. Further, the impact of temperature on the current targeted ECS‐HI system, which usually produces haploids via gamete cell fusion, provides new insights not only into HI but also into the plant reproductive mechanism. Moreover, we concluded that there is no need to create double mutants of ECS1 and ECS2 in rapeseed, as a single ECS1 mutant is as effective as a double mutant in Arabidopsis (Zhao et al. 2025). Along this in future, a detailed mechanistic insight is required to analyze the impact of high temperature on fertilization failure, by observing the fertilization process under different range of temperatures.

Conflicts of Interest

The authors declare no conflicts of interest.

Supporting information

Supporting File

Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request. Moreover, supporting data can be found online in the Supporting section of article.