1 Introduction

Wheat (Triticum aestivum L.) serves as a foundational food crop worldwide, contributing approximately 20% of dietary calories for humanity. Enhancing wheat productivity and adaptability has become essential for tackling worldwide issues like climatic shifts and demographic expansion. Hybrid wheat development stands out as an innovative approach to boost crop performance and stress resilience by utilizing the performance advantages of hybrid offspring. Sterile male plants form the backbone of this strategy, guaranteeing controlled pollination and streamlining hybrid seed generation. Modern innovations, such as genome-based technologies and accelerated inbred line creation (DH methods), have dramatically improved the efficiency of breeding sterile male varieties. These approaches offer reliable solutions for establishing sterility frameworks required in hybrid wheat programs (Li et al., 2020; Huang et al., 2024).

Three main biological pathways induce male sterility in wheat: cell-based sterility (CMS), temperature-light responsive genetic sterility (TPSGMS), and chromosomal sterility. CMS mechanisms, exemplified by the recently discovered AL-type variant, depend on unique mitochondrial gene combinations that disrupt functional pollen development (Hao et al., 2021). TPSGMS models, regulated by climatic factors including heat and daylight duration, are valued for their operational flexibility in hybrid seed generation (Liu et al., 2021). Cutting-edge methods like CRISPR-based gene editing allow targeted modifications of fertility control genes such as Ms1, significantly speeding up the creation of non-fertile plants for hybrid cultivation (Okada et al., 2019).

This study will explore the mechanism of male sterility in wheat, improve the development of sterile lines, and evaluate its effectiveness in the hybrid system. This study aims to discover and analyze new genetic regulatory factors for infertility, design enhanced breeding programs that combine traditional and biotechnological methods, and promote the creation of high-performance wheat hybrids resistant to environmental stress. This study, in combination with recent research, seeks methods to increase wheat yield and shorten breeding time, and addresses the issue of food shortage through innovative agricultural practices. Breakthroughs such as the TPSGMS system and sophisticated gene editing tools have highlighted the paradigm shift capability of modern hybrid wheat research.

2 The Basic Principle of Male Sterility in Wheat

2.1 Definition and classification of male sterility

Male sterility in wheat refers to the inability of plants to produce effective pollen, thereby inhibiting self-pollination and simplifying the production of hybrid seeds (Hernould et al., 1993). This characteristic is classified into three categories: cytoplasmic male sterility (CMS), nuclear gene male sterility (GMS), and temperature-light response genetic male sterility (TPSGMS). CMS is triggered by mitochondrial genomic abnormalities or functional defects, and common genes such as orf279 interfere with pollen formation (Hao et al., 2021). Its stability and compatibility with restorer lines make it the core of hybrid breeding (Yang and Fu, 2024).

GMS is caused by mutations in recessive nuclear genes. The inactivation of key fertility genes (such as Ms1) can induce infertility, and this mechanism has been applied to the gene editing platform (Okada et al., 2019). TPSGMS regulates sterility through the interaction between genes and the environment. For example, specific strains are infertile at low temperatures and restore fertility at room temperature, providing flexible hybridization protocols (Liu et al., 2021). The three types of systems each have their advantages: CMS ensures infertility stability, and TPSGMS does not require maintainers, simplifying the breeding process (Wan et al., 2019).

2.2 Genetic mechanisms of male sterility

Male sterility in wheat stems from the dynamic interaction between the nuclear and mitochondrial genomes. CMS is often triggered by mitochondrial dysfunction. For instance, orf279 causes mitochondrial abnormalities and hinders pollen development. Nuclear recovery genes (such as Rf) can reverse this effect and restore fertility to support hybridization applications (Murai et al., 2016). GMS relies on nuclear gene mutations. For example, CRISPR/Cas9 is used to knock out the key pollen development gene Ms1 to rapidly create sterile lines, highlighting the innovative value of molecular breeding (Okada et al., 2019; Nie et al., 2023).

TPSGMS is regulated by environmental signals. Temperature and light-sensitive genes were differentially expressed under different conditions. Transcriptome studies showed that meiosis and energy metabolism-related genes were significantly downregulated during the infertility stage (Bai et al., 2021). These mechanisms reveal the multi-dimensional regulatory network of infertility and the optimization potential of genetic tools.

2.3 The influence of the environment on the expression of male sterility

Temperature and light are the core regulatory factors of the TPSGMS system. Low temperature inhibits meiosis and glucose transport in TGMS lines, resulting in pollen defects (Liu et al., 2021). Photoperiod regulates the fertility of PGMS, with long-day inhibition and short-day recovery, mediated by the photosensitive gene network (Li et al., 2020; Murai et al., 2016).

Abiotic stresses (such as drought and salt damage) exacerbate infertility by disrupting cellular homeostasis. Anther oxidative damage and metabolic disorders in extreme environments further weaken fertility (Zhang et al., 2018). Precise regulation of environmental conditions can flexibly manage sterile phenotypes and enhance the operability of hybrid wheat production (Ma et al., 2017).

3 Development of Male Sterile Lines in Wheat

3.1 Methods for developing cytoplasmic male sterile (CMS) lines

Mitochondrial-related male infertility (CMS) is caused by maternal inherited mitochondrial DNA mutations or structural variations. The construction of CMS lines requires the continuous backcross between the donor system (carrying CMS cytoplasm) and the reincarnation parent (maintainers) to introduce specific cytoplasmic characteristics into cultivated wheat. For example, Al-type CMS was constructed using mitochondrial abnormalities in Triticum timopheevii wheat, in which the orf279 gene causes infertility by interfering with the regulation of cell death in the trellice layer (Hao et al., 2021). Modern sequencing technology has improved the detection accuracy of CMS-related genes and their regulatory pathways, accelerating the development of sterile lines.

The introduction of wild species (such as jointwheat) can broaden the genetic diversity of CMS. For example, the CMS line derived from Aegilops crassa has photoresponsive sterility characteristics and is suitable for hybrid seed production under specific climates (Murai et al., 2016). CMS remains the mainstream of hybrid breeding due to its stability and compatibility with restorer lines. However, screening for an efficient conservation-recovery combination to maintain sterile cytoplasm remains a challenge. Combining CMS breeding with rapid purebred technology (DH method) can shorten the research and development cycle and achieve rapid trait fixation (Li et al., 2020).

3.2 Approaches to induce nuclear male sterile (NMS) lines

Nuclear gene male sterility (NMS) is caused by mutations in genes related to pollen formation. Traditional methods include chemical mutagenesis (such as EMS treatment) and radiation mutagenesis. CRISPR/Cas9 gene editing has revolutionized the development of NMS. By targeting and knocking out key reproductive genes such as Ms1 (regulating feltine layer function and pollen viability), sterile lines have been precisely created (Singh et al., 2018; Okada et al., 2019).

Temperature-sensitive infertility (TGMS) is another strategy. TGMS is infertile when exposed to low temperature during meiosis and regains fertility at room temperature, simplifying seed reproduction (Liu et al., 2021). Transcriptome analysis revealed that the expression of meiosis and energy metabolism genes was inhibited in sterile plants, clarifying the molecular mechanism (Bai et al., 2021). Integrating traditional breeding with genomic tools has enhanced the development efficiency of NMS (Kim and Zhang, 2018).

3.3 Application of molecular marker-assisted selection in male sterile line development

Molecular marker-assisted selection (MAS) enhances breeding efficiency by precisely tracking sterility related genes. SSR and SNP markers were used to monitor mitochondrial genes (such as orf279) and recovery genes (such as Rf) in CMS lines to ensure cytoplasmic purity and fertility recovery (Mongkolsiriwatana et al., 2019; Hao et al., 2021).

For the NMS line, MAS can rapidly screen sterile alleles such as Ms1, reducing the time consumption for phenotypic identification (Okada et al., 2019). High-throughput sequencing technologies (such as GBS) provide genome-wide marker data, facilitating the mining of new sterile genes and the integration of breeding. MAS can also simultaneously select agronomic traits such as sterility and disease resistance to optimize the breeding of hybrid wheat (Liu et al., 2021).

3.4 Phenotypic and molecular characterization of male sterile lines

Phenotypic analysis of sterile lines includes the assessment of pollen failure rate, anther morphology and environmental stability. Microscopic observation and fertility testing were used to quantify indicators such as pollen failure and seed setting rate (Ding et al., 2018). The CMS system needs to focus on detecting the intergenerational stability of sterility and the compatibility of the restlessness line. Molecular studies have revealed the mitochondrial-nuclear genomic interactions (such as the mechanism of action of orf279) (Hao et al., 2021).

The gene expression profile of NMS needs to be analyzed during the pollen development period. Omics studies have shown that pathways such as glucose transport and cytoskeleton formation are disordered in sterile plants (Bai et al., 2021). Epigenetic analysis (such as DNA methylation) elucidated the regulation of environmental factors on infertility genes (Liu et al., 2021). The integration of phenotypic and molecular data enhances the precise identification of sterile lines and supports the application of hybrid breeding.

4 Utilization of Male Sterile Lines in Wheat Breeding

4.1 Role of male sterile lines in hybrid wheat production

Male sterile lines form the core of hybrid wheat systems by enabling controlled pollination and maintaining seed genetic purity. These lines promote cross-pollination to exploit hybrid vigor, leading to improved yield, stress tolerance, and grain quality in hybrid varieties. For instance, temperature-photoperiod responsive genetic male sterile (TPSGMS) lines are widely adopted in Chinese hybrid programs. Their fertility status can be adjusted through climate manipulation, offering practical solutions for hybrid seed generation under defined conditions (Li et al., 2020). TPSGMS lines developed via accelerated inbred line (DH) methods have shortened breeding timelines while enhancing traits like yield stability and pathogen resistance (Hongsheng et al., 2020).

Beyond seed production, these lines aid in transferring valuable traits from wild or non-adapted germplasm, such as drought resilience or disease immunity. Gene-editing tools like CRISPR/Cas9 allow precise modification of fertility genes, accelerating the design of custom sterile lines for hybrid programs (Okada et al., 2019; Chen et al., 2020). Merging conventional breeding with biotechnological innovations ensures male sterile lines remain vital for maximizing hybrid potential, directly addressing global agricultural demands.

4.2 Establishing compatibility between CMS lines and restorer lines

Successful hybrid systems depend on compatibility between cytoplasmic male sterile (CMS) lines and fertility-restoring lines. Restorer lines carry Rf genes that neutralize mitochondrial-induced sterility, enabling hybrid fertility. Key restorer genes, such as Rf3 located on chromosome 1BS, have been mapped to improve fertility restoration in CMS hybrids (Geyer et al., 2016). Molecular marker-assisted selection (MAS) enhances the identification and integration of these genes, optimizing breeding efficiency (Zhang et al., 2018).

Compatibility also relies on CMS sterility stability and hybrid fertility uniformity. CMS systems involving mitochondrial genes like orf279 demonstrate consistent sterility and broad restorer adaptability (Hao et al., 2021). Wild wheat relatives (e.g., Aegilops) have expanded CMS and restorer genetic diversity, improving adaptability across environments (Murai et al., 2016). Combining phenotypic screening, molecular testing, and gene expression profiling ensures robust hybrid compatibility, advancing breeding program effectiveness.

4.3 Contribution of male sterile lines to genetic resource innovation

Male sterile lines drive genetic innovation by enabling trait transfer and diversifying breeding materials. They allow crossbreeding with wild or exotic germplasm, introducing novel characteristics into elite wheat varieties. For example, Aegilops-derived CMS lines enrich mitochondrial diversity, providing new sterility sources and restorer combinations (Hao et al., 2021). TPSGMS lines further reveal how environmental factors regulate sterility genes, aiding climate-resilient hybrid design (Bai et al., 2021).

Proteomic and transcriptomic analyses of sterile lines have uncovered genes controlling carbohydrate allocation and tapetal function, guiding the development of stress-tolerant, high-yield hybrids. These lines thus serve dual roles: supporting hybrid seed systems and advancing plant biology research. Studies on wheat anther development, including I2-KI staining, highlight temperature’s critical impact on fertility during pollen maturation stages (Zhang et al., 2018) (Figure 1).

Figure 1 Comparison of plant anthers and I2-KI staining in sterile/fertile conditions (Adopted from Zhang et al., 2018)

4.4 Integrating male sterility with advanced technologies such as gene editing

Combining male sterility with gene-editing tools has transformed wheat breeding. CRISPR/Cas9 enables precise disruption of fertility genes like Ms1, essential for pollen wall formation, to rapidly generate sterile lines for hybrid use (Okada et al., 2019). Similar strategies target mitochondrial genes in CMS systems, enhancing sterility reliability.

Genomic selection and network analysis (e.g., gene co-expression studies) identify sterility regulators, optimizing hybrid combinations (Bai et al., 2021). Editing tools also allow stacking sterility with traits like disease resistance, creating multifunctional hybrids. These advances promise next-generation wheat varieties capable of meeting rising food demands under climate challenges.

5 Challenges in Research and Application of Male Sterile Lines

5.1 Current status of male sterile line research globally and domestically

Global research on male sterile wheat lines has achieved notable advancements, particularly in hybrid-focused nations like China, India, and the United States. In China, studies emphasize temperature-light responsive genetic male sterile (TPSGMS) systems, which show high adaptability for hybrid seed development. Transcriptomic investigations, for example, have pinpointed sterility-related genes, refining hybrid breeding accuracy (Yang et al., 2021). Innovations like CRISPR/Cas9 have enabled targeted genetic modifications of fertility genes (e.g., Ms1 and Ms2), accelerating sterile line creation (Zhang et al., 2021).

Regionally, sterile line utilization reflects local priorities. European programs prioritize cytoplasmic male sterile (CMS) lines from wild species like Aegilops, enriching hybrid genetic diversity (Li et al., 2019). However, developing regions face barriers such as limited access to advanced tools and funding. Hybrid wheat adoption in Sub-Saharan Africa, for instance, is hindered by inadequate seed production infrastructure. Strengthening global collaboration through shared genomic resources and technology transfer could address these gaps.

5.2 Technical barriers in the widespread application of male sterile lines

Multiple technical hurdles limit sterile line adoption. A primary challenge is maintaining sterility consistency across diverse climates. Research on temperature-sensitive lines (e.g., Bainong series) demonstrates how shifting temperatures disrupt sterility, compromising hybrid reliability (Su et al., 2018). CMS systems face complexities in balancing mitochondrial sterility genes (e.g., orf279) with nuclear restorer genes, which vary across genetic backgrounds (Hao et al., 2021). Additionally, hybrid seed production remains costly due to labor-intensive maintenance and multiplication.

Emerging technologies like CRISPR/Cas9 face regulatory uncertainties in many regions, limiting their scalability (Zhou et al., 2019). Standardized protocols for sterility trait evaluation are also lacking, delaying breeding progress. Overcoming these challenges demands coordinated efforts to develop climate-resilient lines, reduce costs via mechanization, and establish universal evaluation frameworks.

5.3 Research needs addressing environmental adaptability and climate change

Climate instability threatens sterile line performance, especially in TPSGMS systems. Proteomic studies reveal that heat stress disrupts sugar metabolism and tapetal function in temperature-sensitive lines, destabilizing sterility (Zhang et al., 2018). To address this, studies must focus on designing climate-resilient sterility mechanisms by exploring wild germplasm genes or mapping stress-responsive regulatory networks (Yang et al., 2021). Integrating predictive models (e.g., GWAS) could identify genetic markers linked to climate tolerance, aiding resilient hybrid development.

Multi-environment field trials are essential to assess sterility stability under extreme conditions, simulating future climate scenarios. Combining sterile lines with precision agriculture tools and stress-hardy pollinator lines can further enhance adaptability. Cross-disciplinary collaboration-spanning genomics, ecology, and agronomy-is vital to ensure sterile lines remain effective amid changing climates.

6 Case Study: Successful Application of Male Sterile Lines in Wheat Hybrid Breeding

6.1 Research design and selection of test lines

Developing and testing male sterile lines for hybrid wheat requires systematic experimental designs combining diverse germplasm and modern technologies. A recent project assessed temperature-light responsive genetic male sterile (TPSGMS) lines for hybrid potential. Researchers crossed two commercial TPSGMS lines with eight restorer lines using a line × tester framework (Abdelkhalik et al., 2019), identifying superior hybrids with high combining ability. Accelerated inbred line (DH) methods stabilized sterility traits in early generations, generating 24 elite lines with stable sterility, disease resistance, and optimized growth (Li et al., 2020).

Molecular tools like marker-assisted selection (MAS) and genomic prediction streamlined the identification of sterility-linked loci, improving breeding precision (Geyer et al., 2016). Phenotypic screening combined with molecular analysis ensured selection of lines with stable sterility and hybrid vigor. CRISPR/Cas9 editing of Ms1 further enabled rapid creation of sterile hexaploid lines for hybrid seed systems (Okada et al., 2019) (Figure 2).

Figure 2 Inheritance and segregation of targeted mutations and male sterility generated with gRNA LTPG1-2 (Adopted from Okada et al., 2019) Image caption: (a) Crossing of the transgenic T0 mutant line GL353-119 with wild-type cv. Gladius. (b) Representative examples of T1 progeny derived from the cross shown in (a). The T1 progeny were selfed to produce T2 seeds. (c) Selection of DsRed-negative (presumed non-transgenic) T2 seeds produced by line T1-1. Scale bar = 1 mm. (d) Representative examples of T2 progeny grown from the selected DsRed-negative seeds. (e) Genotyping of the 26 DsRed/Cas9-negative T2 progeny. The +1 mutant allele contains an AluI restriction site that is not present in the WT allele. Cleavage at the AluI restriction site results in a 210 bp band (red arrow head) (Adopted from Okada et al., 2019)

6.2 Implementation of male sterile lines in field breeding trials

Field testing validates sterile line performance under practical conditions. Multi-location trials evaluated TPSGMS hybrids paired with high-yield restorers, demonstrating adaptability and seed production efficiency (Li et al., 2020). These lines achieved over 95% seed purity under optimal pollination. Similarly, CMS lines derived from Aegilops cytoplasm were tested with Rf3-carrying restorers, showing reliable fertility restoration and hybrid vigor (Geyer et al., 2016).

Mechanized pollination systems improved large-scale seed production efficiency. Enhanced floral traits (e.g., anther extrusion) increased cross-pollination rates, reducing labor costs (Boeven et al., 2018).

6.3 Analysis of yield, stability, and hybrid vigor outcomes

Hybrid success hinges on yield gains, environmental stability, and vigor expression. TPSGMS hybrids outperformed conventional varieties by 15–20% in yield, with enhanced resistance to yellow rust and powdery mildew (Li et al., 2020). Vigor was evident in traits like spike size and grain weight-hybrids from TPSGMS lines K456s and K78s excelled in five of nine yield traits (Abdelkhalik et al., 2019; Khlaimongkhon et al., 2019).

MAS-optimized genetic combinations boosted hybrid performance. Long-term trials confirmed economic viability through efficient seed production and reduced inputs. These cases demonstrate the transformative role of sterile lines in advancing hybrid wheat breeding.

7 Suggestions for Promoting the Research and Utilization of Male Sterile Lines

7.1 Cooperative research on promoting the development of male sterile lines

To promote the research on male sterile lines, it is necessary to strengthen the collaboration among universities, agricultural research institutions and seed enterprises. Integrating multidisciplinary resources such as genetics and molecular biology can significantly improve the breeding efficiency of hybrid wheat. For instance, the recessive infertility gene ms1s was successfully located through collaborative genetic research, verifying the value of multi-party collaboration in exploring infertility resources (Yang et al., 2021). Cross-border cooperation is particularly important for addressing regional challenges. For instance, the wheat breeding project based on CMS in Egypt demonstrated the advantages of combining global experience with local demands by screening conservation and restoration lines adapted to the local environment (El-Rady and Soliman, 2023).

Establishing a global germplasm resource sharing platform and open databases (such as sterile gene banks) can accelerate the research and development process of sterile lines. Such platforms can promote data intercommunication and technology sharing, ensuring that global researchers jointly participate in technological innovation.

7.2 Strengthen support for the integrated breeding project of male sterile lines

The intensified breeding project requires increased investment in infrastructure, technical tools and talent cultivation. Chinese studies have shown that the accelerated pure line (DH) breeding technology can efficiently cultivate stable temperature and photosensitive sterile lines (TPSGMS) and shorten the research and development cycle (Li et al., 2019). Promoting such technologies to other regions will significantly enhance breeding efficiency. The government and the private sector should give priority to funding hybrid wheat projects, and support field trials and mechanized seed production through special funds to break through the bottleneck of large-scale production.

For instance, in the study of the CMS system in Egypt, mechanical pollination increased the seed production efficiency by 30% (El-Rady and Soliman, 2023). In addition, conducting training on molecular breeding techniques (such as CRISPR/Cas9) can enhance the technical proficiency of breeders. Recent cases of precise editing of genes such as Ms45 have confirmed the potential of genetic tools in constructing hybrid adaptive sterile lines (Singh et al., 2018).

7.3 Enhance farmers' awareness and adoption of male sterile line technology

The recognition and acceptance of farmers are the key to the implementation of technology. The promotion should focus on the yield-increasing and stress-resistant advantages of hybrid wheat. The field demonstration of hybrid varieties based on CMS and TPSGMS shows that their yield is 15-20% higher than that of conventional varieties, which is highly attractive (Abdelkhalik et al., 2019). Through participatory breeding, farmers can select hybrid varieties that are adapted to the local climate and meet quality requirements, which can enhance technical adaptability. Providing seed subsidies in the initial stage can reduce the adoption cost.

Incorporate the technical principles of sterile lines into the agricultural technology training system, expand publicity in combination with digital platforms and grassroots promotion stations, especially benefiting remote areas (Okada et al., 2019).

8 Concluding Remarks

Research on male sterile lines has significantly promoted the development of wheat breeding, especially in the field of hybrid seed production. The development of temperature-photoperiod-sensitive sterile lines (TPSGMS) with strong environmental adaptability is an important direction. Their field performance combines stable sterility and high-yield potential, supporting large-scale application. Rapid pure line (DH) technology and gene editing tools (such as CRISPR/Cas9) have accelerated the creation of high-quality sterile lines - for example, knockout of the Ms1 gene can rapidly induce infertility and significantly shorten the research and development cycle. Proteomics has revealed the molecular mechanisms of pollen failure, such as energy metabolism imbalance and abnormal glucose transport. These systems not only optimize hybrid seed production but also broaden the genetic diversity of wheat, which is crucial for ensuring global food security.

The application of male sterility technology has revolutionized the wheat hybridization system, efficiently converting heterosis into yield gains. The field yield of hybrid varieties based on TPSGMS reached 20%, and the disease resistance (such as stripe rust and powdery mildew) was significantly improved (Abdelkhalik et al., 2019). However, large-scale production still needs to break through the bottlenecks of pollination efficiency and cost. Mechanical pollination and molecular marker-assisted selection (MAS) have shown potential. For example, optimizing the structure of flower organs can increase the cross-pollination rate (Boeven et al., 2018). Successful cases show that integrating traditional breeding with molecular technology is the key to addressing agricultural challenges.

Future research needs to focus on the climate adaptability of sterile lines, especially their ability to cope with extreme weather. The exploration of new genetic resources in wild relatives and the analysis of new mechanisms of infertility (such as mitochondrial-nuclear gene interactions) can break through the existing limitations. Genomic technologies (such as genome-wide association analysis) and epigenetic studies (such as DNA methylation regulation) will deepen the understanding of the molecular network of infertility. For instance, the temperature-regulated methylation mode of TPSGMS provides a new idea for heat-resistant design (Liu et al., 2021). In addition, it is necessary to strengthen the technical training for farmers, demonstrate the yield-increasing benefits of hybrid varieties through demonstration fields, and build a cooperation network among the government, enterprises and scientific research institutions to promote the implementation of the technology.

Acknowledgments

We would like to thank the research team for their suggestions on my manuscript.

Conflict of Interest Disclosure

The authors affirm that this research was conducted without any commercial or financial relationships that could be construed as a potential conflict of interest.

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