Male Sterility Systems in Hybrid Rice Development for Abiotic Stress Tolerance: A Systematic Review of Genetic Sources, Molecular Mechanisms, and Breeding Strategies

3.1 Keyword Co-occurrence and temporal research trends

All literature included in this review met the eligibility criteria presented in Table 1 and comprised studies published between 2017 and 2026 on environmental stress tolerance in hybrid rice. Bibliometric visualisation using VOSviewer identified two major thematic clusters ( Figure 2): drought-related studies associated with CRISPR/Cas9, agronomic traits, and homozygosity, and more recent heat-stress research linked to metabolic regulation, catalase activity, and hydrogen peroxide scavenging. Node size indicated keyword frequency, with genetics, phenotype, drought stress, and gene ontology emerging as the most dominant themes. At the same time, network density highlighted strong co-occurrence among genetics, phenotype, environmental stress, and genetic engineering. Temporal overlay analysis further revealed a shift in research focus from foundational topics such as hybridisation, QTL mapping, genomics, and combining ability (2018) toward drought tolerance, CRISPR/Cas9, and heat stress (2020), followed by recent emphasis (2022–2026) on Rf3 genetics, gene expression, enzymology, antioxidant regulation, and high-temperature tolerance. Overall, these trends indicate a shift from conventional breeding toward molecular and physiological strategies to improve antioxidant defence systems and heat resilience in hybrid rice.

Fig 2. Distribution of articles by abiotic stress.

3.2 Diversity and functional architecture of male sterility systems in hybrid rice breeding

Hybrid rice (Oryza sativa) performance is strongly influenced by Cytoplasmic Male Sterility (CMS) systems, particularly Wild-Abortive CMS (WA-CMS), which are regulated by mitochondrial genes that disrupt pollen development and enable efficient hybrid seed production. Beyond reproductive control, CMS systems contribute to agronomic improvement through enhanced specific combining ability (SCA), heterosis, and hybrid vigor arising from interactions among CMS, maintainer, and restorer lines. CMS-derived hybrids also exhibit superior germination, stronger root architecture, and improved seedling establishment under drought stress, reflecting the importance of nuclear–cytoplasmic interactions and stress-responsive metabolic regulation.

As summarized in Table 2, hybrid rice breeding over the last decade has been predominantly centred on CMS-based three-line systems, with WA-CMS remaining the most widely utilized platform because of its stable sterility expression, broad fertility restoration capacity, and strong association with high yield performance (Hong-Bing et al., 2017; Widyastuti et al., 2017; Liao et al., 2019; Luo et al., 2019; El-mowafi et al., 2021; Madhusudan et al., 2022; Awad-allah et al., 2022; Li et al., 2023; Ghazy et al., 2023; Bhuyan et al., 2025). The recurrent use of elite CMS lines such as IR58025A and IR69625A further indicates the continued dependence of breeding programs on a limited number of reliable cytoplasmic donor sources. At the molecular level, CMS is associated with aberrant mitochondrial genes that induce maternally inherited male sterility, providing an efficient biological system for large-scale hybrid seed production without manual emasculation.

Despite these advantages, several constraints continue to limit the long-term sustainability of CMS-based breeding. Narrow cytoplasmic diversity increases vulnerability to environmental and biological stresses, while sterility expression in certain CMS backgrounds remains sensitive to temperature and photoperiod fluctuations (Liu et al., 2021; Zhang et al., 2021; Chen et al., 2021; Lin et al., 2023). Another major challenge is the requirement for compatible nuclear restorer-of-fertility (Rf ) genes to achieve complete fertility recovery (Nabi et al., 2022; You et al., 2023; Wei et al., 2024; Wang et al., 2025). Consequently, recent research has increasingly shifted toward improving the efficiency of fertility restoration, diversifying restorer germplasm, and elucidating the genomic regulation of nucleus–cytoplasm interactions (Anis et al., 2018; Hamad et al., 2023; Nagaraju et al., 2023; Wei et al., 2024; Wang et al., 2025; Niu et al., 2025).

The strategic importance of restorer lines is evident from the repeated use of elite genotypes such as KMR, Guihui5501, Chenghui727, and Zhonghui9308, which exhibit stable fertility restoration and superior performance across multiple CMS backgrounds. However, the repeated deployment of these lines also indicates that current breeding programs still rely on a relatively narrow pool of elite donor germplasm. At the genetic level, fertility restoration is primarily regulated by nuclear-encoded Rf genes that suppress sterility-associated mitochondrial transcripts, representing a clear example of coordinated nucleus–cytoplasm coevolution in crop breeding systems (Anis et al., 2018; Nabi et al., 2022; Nagaraju et al., 2023; Wei et al., 2024; Wang et al., 2025).

To improve breeding efficiency, recent programs increasingly integrate molecular markers, high-throughput genotyping, and genomic selection to rapidly identify and develop superior restorer lines. These advances reflect the broader transition from conventional phenotype-based selection toward genomics-assisted hybrid breeding to enhance stress resilience, fertility stability, and sustainable yield performance under changing environmental conditions.

3.3. Physiological and molecular mechanisms of stress tolerance in hybrid Rice

CMS-based hybrid rice exhibits integrated physiological and molecular adaptations that enhance tolerance to major abiotic stresses, including drought, heat, salinity, nutrient deficiency, and low temperature ( Figure 3). Physiological responses mainly involve improved water-use efficiency, carbohydrate regulation, and antioxidant defense systems (Zou et al., 2018; Liao et al., 2019; Yang & Zhang, 2020; Chen et al., 2021; Ghazy et al., 2023; Li et al., 2023; Wang et al., 2025). Under drought stress, SRL1/SRL2 mutants reduce stomatal density, conductance, and transpiration, thereby minimizing water loss (Liao et al., 2019). Hormonal treatments, such as kinetin and jasmonate, further improve chlorophyll retention, osmotic adjustment, and spikelet hydration under drought and heat stress (Yang & Zhang, 2020; Ghazy et al., 2023).

Fig 3. Mechanisms of stress tolerance in CMS-based hybrid rice.

Stress adaptation is also supported by maintenance of carbohydrate homeostasis and enhanced antioxidant activity. The sterile line Dexiang 074A maintained soluble sugar content and panicle dry matter allocation under drought conditions, while the restorer line R996 preserved soluble sugars and starch reserves during heat stress, supporting osmoprotection and metabolic stability (Li et al., 2023; Zhou et al., 2025). Increased activities of ascorbic acid (AsA), superoxide dismutase (SOD), catalase (CAT), peroxidase (POD), and ascorbate peroxidase (APX) reduce ROS accumulation and oxidative damage under stress conditions (Liao et al., 2019; You et al., 2023; Wang et al., 2025). Application of methyl jasmonate (MeJA) further strengthens antioxidant defense by enhancing AsA and CAT activity, thereby protecting reproductive tissues from heat-induced injury (Chen et al., 2021).

At the molecular level, stress tolerance is regulated through gene expression and QTL introgression. The DRO1 gene promotes deep root development under drought stress, while qDTY1.1, qDTY12.1, and qSDT12–2 contribute to maintaining plant architecture and yield stability under water limitation (Luo et al., 2019; Nabi et al., 2022; Nagaraju et al., 2023). Heat tolerance is strongly associated with heat shock proteins (HSPs), including OsHSP1, cHsp70–1, OsHsp17.9A, and OsHSP60-3B, which stabilize proteins and reduce oxidative damage, thereby preserving pollen viability under high temperatures (Lin et al., 2023; Zhou et al., 2025).

Under salinity stress, the qDYST-1 locus and SKC1NB allele regulate transcriptional responses and Na + transport to reduce ion toxicity (Ding et al., 2022; Niu et al., 2025). Tolerance to phosphorus deficiency is associated with qRN5, qRDW5, qRL6, and Pup1, which improve root development and phosphorus uptake efficiency (Anis et al., 2018; Madhusudan et al., 2022). Cold tolerance remains comparatively underexplored, although the Cold1 gene has been identified as a regulator of low-temperature sensing through Ca2 + –dependent signaling pathways involving RGA1 (Wei et al., 2024). Overall, these findings demonstrate that modern CMS-based hybrid rice breeding increasingly integrates physiological adaptation with molecular and genomic regulation to enhance stress resilience and yield stability under adverse environments.

3.4 Relationships between abiotic stress, adaptive traits, and yield performance in hybrid Rice

CMS-based hybrid rice exhibits coordinated morphological, physiological, and reproductive adaptations to abiotic stress ( Figure 4), with drought and heat stress representing the most extensively studied constraints. Drought tolerance is primarily associated with enhanced root architecture, seminal root length, biomass accumulation, and seedling vigor, which collectively improve water uptake and sustain vegetative growth under limited water availability (Widyastuti et al., 2017; Hong-Bing et al., 2017; Liao et al., 2019; Luo et al., 2019; Nabi et al., 2022; Ghazy et al., 2023). In contrast, heat stress predominantly disrupts reproductive processes, including spikelet fertility, pollen viability, another development, and seed set formation, with spikelet fertility consistently identified as the principal determinant of yield stability under high-temperature conditions (Zhang et al., 2017; Chen et al., 2021; Lin et al., 2023; You et al., 2023; Wei et al., 2024; Zhou et al., 2025). Salinity, phosphorus deficiency, and cold stress remain comparatively less explored but are generally linked to changes in root development, biomass accumulation, seedling vigor, and reproductive performance (Anis et al., 2018; Thummala et al., 2022; Niu et al., 2025).

Fig 4. Conceptual framework of stress-adaptive traits associated with grain yield and breeding performance in CMS-based hybrid rice.

Despite these adaptive responses, physiological tolerance does not always translate into superior productivity, indicating a complex relationship between stress-resilient traits and agronomic performance. Several drought-tolerant hybrids with improved germination and root traits, for example, did not consistently achieve high grain yields, while maintaining reproductive viability under heat stress was sometimes insufficient to sustain seed production (Widyastuti et al., 2017; Chen et al., 2021). In addition, stress-responsive traits are frequently governed by non-additive gene action, reflected by high specific combining ability (SCA) and strong heterotic effects, emphasizing the importance of parental genetic interactions and optimal hybrid combinations in the development of climate-resilient hybrid rice (Widyastuti et al., 2017; Pang et al., 2017).

3.5 Breeding strategies for developing stress-tolerant hybrid rice

Based on the reviewed studies, hybrid rice breeding strategies are increasingly directed toward developing abiotic stress-tolerant genotypes while maintaining high yield potential. As shown in Figure 5, the three-line system remains the dominant breeding approach because of its stable male sterility and well-established seed production system. However, its effectiveness is constrained by the limited availability of compatible restorer lines, leading recent breeding efforts to focus on improving both fertility restoration and stress tolerance simultaneously (Chen et al., 2021). Restorer lines, therefore, serve not only as fertility recoverers but also as important sources of adaptive traits under stress conditions (Sravanraju et al., 2024; Wei et al., 2024; Bhuyan et al., 2025). Drought-tolerant restorers were reported to maintain fertility and productivity under water-deficit conditions (Nagaraju et al., 2023), while Guihui5501 combined high yield, grain quality, and cold tolerance (Wei et al., 2024).

Fig 5. The relationship between breeding strategies and approaches to developing hybrid rice tolerant to abiotic stress.

The development of stress-tolerant hybrids is also strongly influenced by parental combining ability. General combining ability (GCA) and specific combining ability (SCA) analyses are widely used to identify superior hybrid combinations under stress environments. Several studies reported strong SCA effects for drought-tolerant hybrids rice (Widyastuti et al., 2017; El-mowafi et al., 2021; Awad-allah et al., 2022), whereas IL50–13 was identified as a promising salinity-tolerant restorer with favorable combining ability for yield traits (Thummala et al., 2022). These findings indicate that non-additive genetic interactions between parental lines largely govern stress tolerance in hybrid rice.

To accelerate breeding efficiency, molecular approaches are increasingly integrated into hybrid rice improvement programs. Marker-assisted selection targeting fertility restoration genes such as Rf3 and Rf4 has been widely applied for identifying stress-tolerant parental lines (Priyadarshi et al., 2025), while QTL introgression remains the primary strategy for improving tolerance to drought, salinity, heat, and phosphorus deficiency (Anis et al., 2018; Luo et al., 2019; Abdelrahman et al., 2021; Bhuyan et al., 2025; Niu et al., 2025). More advanced technologies, including whole-genome sequencing and CRISPR/Cas9-based genome editing, are further enhancing the precision of stress-tolerance improvement and accelerating the development of resilient hybrid rice varieties adapted to changing environmental conditions (Thummala et al., 2022; Sheng et al., 2023).

3.6 Challenges and future perspectives

Although significant progress has been made in developing abiotic stress-tolerant hybrid rice through CMS-based systems, maintaining stable sterility expression and fertility restoration under stress conditions remains a major challenge, particularly under high-temperature stress, which severely disrupts reproductive development and spikelet fertility (Widyastuti et al., 2017; Chen et al., 2021). Most existing studies also focus on single-stress tolerance, whereas combined-stress responses and environmental stability remain insufficiently explored. Pang et al. (2017). demonstrated the potential of DMS-assisted recurrent selection for developing drought- and salinity-tolerant lines, although evaluations were still conducted under limited environmental conditions.

Another key limitation is the weak integration between molecular breeding and agronomic management. Molecular studies largely emphasise QTL mapping, introgression breeding, and candidate gene identification for stress tolerance (Anis et al., 2018; (Thummala et al., 2022), whereas agronomic approaches, such as growth regulator applications, are generally investigated separately (Ghazy et al., 2023; Hamad et al., 2023). This indicates the need for more integrated strategies that combine molecular genetics, plant physiology, and crop management to simultaneously improve stress tolerance, yield stability, and hybrid seed production efficiency.

Future progress in stress-tolerant hybrid rice breeding will likely depend on the utilisation of novel germplasm and advanced genomic technologies. Wild rice species such as Oryza rufipogon provide valuable sources of adaptive alleles for drought and salinity tolerance (Luo et al., 2019; Thummala et al., 2022), while whole-genome sequencing and genome editing offer opportunities for precise identification and transfer of stress-responsive genes. These advances could accelerate the development of climate-resilient hybrid rice that maintains fertility, productivity, and stability under increasingly complex environmental conditions. Accordingly, future research should prioritise multi-location and multi-season evaluations, combined-stress analyses, and the development of environmentally stable sterility systems to support sustainable hybrid rice production under climate change.