Exploring the Strategies of Male Sterility for Hybrid Development in Hexaploid Wheat: Prevailing Methods and Potential Approaches

Abstract

Hybrid breeding has emerged as a pivotal strategy to enhance wheat crop yield, a critical step to meet the escalating food demand for the growing global population. Heterosis in wheat can boost crop yield; however, harnessing heterosis in bread wheat is complex and hindered by the species' inherent tendency for self-pollination, high genome ploidy, and limitation of male sterile lines. In contrast, the availability of genetic male sterility, and altering reproductive biology such as anther extrusion and floret opening, is challenging but could facilitate outcrossing. Despite the advancements in sterility systems and molecular tools, an efficient and environmentally stable wheat hybrid production system is still lacking. In this review, we examine the advantages and limitations of different male sterility sources utilized to date including, chemical hybridizing agents (CHAs), cytoplasmic male sterility (CMS), nuclear genic male sterility (NGMS), and environmental-sensitive male sterility (ESMS). Furthermore, we explore the potential of molecular tools such as marker-assisted selection (MAS), genome editing, and other genetic engineering approaches to accelerate hybrid wheat breeding efforts. Future research directions are proposed to develop robust, cost-effective systems by integrating conventional and molecular approaches with advanced screening methods including cytogenomics and next generation sequencing (NGS), which can reliably help to produce stable, high-yielding, and resilient hybrid wheat cultivars compared to current open-pollinated varieties. Collectively, these efforts are vital to achieve the food demands for escalating population under climate change scenario.

Hexaploid Wheat and its Significance as Hybrid Wheat

Hexaploid wheat is distinct in its evolution from other cultivated cereals and it is (2n = 6X = 42) consisting of three genomes (AABBDD) derived from three progenitor species resulting from at least two spontaneous hybridization events (Rosyara et al. 2019; Qi et al. 2007; Sakuma et al. 2019; Matsuoka and Nasuda 2004). Further, the haploid nucleus of hexaploid wheat (AABBDD) consists of almost 17 billion base pairs, including 80% of repetitive sequences, 19% (introns, regulatory sequences, and noncoding RNA), and only 1% of the genome consists of coding genes (Nielsen et al. 2014; Bennett 1972; Consortium et al. 2018). Being domesticated about 10,000 years ago, hexaploid bread wheat contributes about 95% of total wheat cultivation worldwide (Dadrasi et al. 2023). It is one of the most widely grown crops with China, India, and the Russian Federation being the top three producers of the globe (Dadrasi et al. 2023). Wheat provides the essential components of the human diet being a source of essential amino acids, minerals, and micronutrients including iron, zinc, and selenium respectively (Tadesse et al. 2019; Mann et al. 2015).

In the last two decades, changes in the living styles and food habits of people have increased the annual wheat demand across the globe with an average rate of 5.8% in East and South Africa, 4.7% in Central and West Africa, 4.3% in Pacific and South Asia, 2.2% in North Africa, 2.2% in Australia and 5.6% in Central Asia (Tadesse et al. 2019; Shiferaw et al. 2013). The global population will escalate to approximately 8.5 billion by 2030. This upward trend suggests a population of 9.7 billion by 2050 and 10.4 billion by the year 2100 (FAO. 2022; Buettner 2022). The prevalence of hunger is expected to be more severe as reports indicate that approximately 770 million people have suffered from malnutrition in 2021 (FAO. 2022). As agricultural land is finite and being further squeezed by human activities and urbanization, to meet global targets, a continuous yield improvement of crop species is required (Tilman et al. 2011; Boeven et al. 2016b). In the past, the green revolution has made a significant contribution to eradicating global hunger, yet wheat production is facing challenges due to unexpected and unwanted weather and the increasing world population. Despite being the crop with the highest acreage in the world, its production still lags behind rice and maize (Tilman et al. 2011). In the light of current challenges, the adoption of hybrid wheat production can emerge as a pivotal strategy to bolster global food security (Selva et al. 2020; FAO. 2022). However, it has been difficult to exploit hybrid wheat due to its higher ploidy, floral structure, lack of efficient male sterility system, impractical fertility control system, and more land area (El Hanafi et al. 2022; Comai 2005). It has been suggested that novel ways should be discovered to identify heterosis and male sterile systems for gaining remarkable success in hybrid wheat (El Hanafi et al. 2022). The area under hybrid wheat has increased up to 0.2% in the last two decades (Longin et al. 2012); however, there is potential to increase more by involving a few more stakeholders. Wheat hybrids produced up to a difference of 2–3 tons per hectare, an increase from 10 to 15% when compare to commercial cultivars (Table 1) with improved grain and straw quality, better fertilizer response, lodging resistance, disease resistance, root penetrance, and grain filling rate/duration (Kempe and Gils 2011; Yang et al. 2022; Longin et al. 2013; Langemeier 2024).The primary goal of this review is to explore traditional and molecular approaches for hybrid development in wheat and to explain the different male sterility procedures for the commercialization of hybrid wheat to meet future food demands.

Table 1.

Yield comparison of hybrid wheat with common wheat across the globe

Country	Common wheat yield (t/ha)	Hybrid wheat yield (t/ha)	References
Germany, Poland, France (Western Europe)	7.0–8.0	9.2	Langemeier (2024)
Australia	2.5	2.8	Hoffman (2024)
India, Pakistan (South Asia)	3.2–3.5	3.8–4.0	Langemeier (2024)
Ukraine, Russia (Eastern Europe)	3.5–5.0	4.5–5.5	Hoffman (2024)
Canada, USA (North America)	3.0–4.0	4.0–4.6	Bruns and Peterson (1997)

Desired Floral Biology for Hybrid Wheat Breeding

Cross-fertilization, a requirement of a hybrid seed production system in wheat, has been observed to be 6.05% in various studies. However, the compact floral architecture and cleistogamy in self-pollinated wheat impose problems by hindering the cross-fertilization of male sterile lines with a diverged pollen source (Martin 1990; Singh 2006). The important floral traits that influence cross-pollination in wheat include stigma size, stigma receptivity, style length, anther size, pollen number, anther extrusion, filament length, and pollen viability (Hucl 1996; Boeven et al. 2016a; Langer et al. 2014; Okada et al. 2019).

Ideally, a male parent for hybrid seed production in wheat should have a higher degree of floret opening, anther extrusion for pollen shedding, higher pollen viability, and availability of wind to disperse the heavy pollen from male to female lines (Fig. 1 (Okada et al. 2018)). Pollen dispersal is observed to be positively associated with plant height, anther size, and length of filament (Joppa et al. 1968). Increased pollen mass, pollen viability, and anther extrusion are shown to be the most promising traits for the selection of male plants (De Vries 1974; Boeven et al. 2016a, 2018). Wide genetic diversity and availability of high heritability for pollen size, anther extrusion, and duration of floret openness have been reported in hexaploid wheat (Langer et al. 2014).

Fig. 1.

Ideal floral biology and plants for hybrid development in wheat. A Ideally female plant (male sterile line) should be male sterile and relatively smaller in height than the male fertile plant to capture the pollen through wind dispersal from taller pollen sources (male fertile line). B Further, female florets should have increased stigma size and prolonged receptivity for capturing the pollens, having higher density and increased length of stigmatic hairs. In parallel, male florets should have a higher degree of floret opening, anther extrusion for pollen shedding, higher pollen viability, and availability of wind to disperse the heavy pollen from male to female lines

An ideal female plant for hybrid seed development in wheat should be male sterile or self-incompatible with increased stigma size and prolonged receptivity for capturing the pollens, having higher density and more increased length of stigmatic hairs (Fig. 1 (Gupta et al. 2015)). In wheat, a spontaneous mutation has given rise to a line with three pistils and is conferred by a single recessive gene (Duan et al. 2015). But this trait may result in decreased seed size and low germination in a hybrid seed production system (Guo et al. 2019; Peng et al. 2008). Moreover, lodicules play a key role in the hybrid breeding system because they help in anther extrusion and pollen capture. Lodicules, filaments, and style exert pressure at the junction of the lemma and palea, such that the angle of the floret opening increases, protruding the anthers outside the floret (Murai et al. 2002b). Large lodicule, soft lemma and palea with spaced spikelets are also important traits for hybrid seed development (Murai et al. 2002a). Synchronization of flowering time between male and female plants is required which can be achieved by exploring the genes responsive to vernalization and photoperiod. Furthermore, the female lines would need to be relatively smaller in height than the male lines to capture the pollen from taller pollen sources. This factor can be controlled by exploiting the height-reducing Rht genes, introduced in wheat during the Green Revolution era (Boeven et al. 2016a; De Vries 1973; Langer et al. 2014; Borojevic and Borojevic 2005).

Availability of Heterosis in Hexaploid Wheat

Crop hybrids should exhibit enhanced biomass, uniform vegetative growth, improved flowering, and a resilient root system (Singh et al. 2023). Heterosis in wheat was observed for the first time in 1919 by Freeman for plant height and subsequently for grain yield in 1934 (Briggle 1963). Focus on mid and better-parent heterosis for grain yield in bread wheat has been emphasized for more than a century (Pal and Alam 1938). The self-pollinating nature of bread wheat made it crucial for the exploitation of hybrid vigor (Sharma et al. 1986). Several studies in the past have depicted that wheat hybrids outperform common wheat varieties for grain yield as well as against different biotic and abiotic stress tolerance (Longin et al. 2013; Hayes 1993). Significant heterosis value of 10.7% was observed for grain yield, susceptibility to frost and leaf rust subsequently (Longin et al. 2013). A study estimated that the exploitation of commercial heterosis to find the right parental combinations, improving the understanding of gene interactions, heritability, and subsequent inbreeding depression is important to start any hybrid breeding program (Adugna et al. 2004).

Conventional Breeding Approaches to Develop Male Sterility in Wheat

Male sterility means that plants are unable to produce viable pollens and hence are incapable of fertilizing the female gametes (Gautam et al. 2023; Belliard et al. 1979). Approaches like hand emasculation, utilization of chemical hybridizing agents (CHAs), cytoplasmic male sterility (CMS), genetic male sterility (GMS), and environmentally controlled male sterility have been utilized for induction of male sterility in hexaploid wheat (Wells and Caffey 1956; Chase and Gabay-Laughnan 2004; Edwards 1983; Jan et al. 1976; Zhou and Wang 2007; Murai et al. 2008; Song et al. 2013). Here, we compared the major challenges of these approaches one by one.

Hand Emasculation

Hand Emasculation is the removal of anther filaments from the floret to avoid self-pollination and to prepare the plant as a maternal line (Florell 1934). Hand emasculation is the most common method that is utilized to develop hybrids for basic studies like measurements of heterosis, combining ability and heritability analysis (Titan et al. 2013; Almutairi 2022; Noorka et al. 2013). Another modified form of hand emasculation is hot water treatment in which a spike of the plant is dipped for 4 min at 43 °C in hot water (Hussain et al. 2012). However, both approaches were not found successful on a commercial scale because these approaches were time-consuming, expensive, labor-intensive and some of the florets remained fertile in the spike (Inagaki et al. 1997).

Chemical Hybridizing Agents (CHAs)

Historically, different chemicals have been utilized to cause male sterility in various crops since the 1950s (Moore 1950; Naylor 1950; Hoagland et al. 1953). In early experiments of wheat hybrid seed production, female plants are treated with gametocides before anthesis (Schulz et al. 1993; Hakraborty and Devakumar 2006). Pollination of male sterile lines is facilitated by wind and obtained F₁ hybrid showed restored fertility (Kempe and Gils 2011). Later, various chemical hybridizing agents (CHAs) have been explored to enhance hybrid wheat breeding programs. These agents include antiauxins, auxins, halogenated aliphatic acids, gibberellins, ethephon, DPX-378, arsenicals, and various patented compounds. These CHAs were tested to improve the efficiency and success in hybrid wheat production (Mcrae 1985).

However, certain drawbacks were identified over time, leading to subsequent improvements in the use of CHAs. These were found to be highly phytotoxic, caused female sterility, and demonstrated an inadequate level of male sterility in selected populations (Rowell and Miller 1971; Jan et al. 1974, 1976; Miller and Lucken 1977). Ideally (i) CHA should induce male sterility in the flowers without impairing female fertility and should be effective for early and late-maturing spikes. (ii) Moreover, it must require a single treatment and should be resilient under adverse weather conditions (Hayes 1993). (iii) Additionally, a CHA should not affect the quality, vigor, or germination rate of F₁ seedlings. (iv) Its effectiveness should bebroad among different purelines/inbreds and should not cause phytotoxicity or mutagenicity in parent plants or their progeny (Kempe and Gils 2011; Hayes 1993).

A variety of gametocides have been evaluated in hybrid wheat production, but currently, only a few are in use (Table 2). Notably, Hybrex, developed by Rohm & Haas, Sintofen (also known as Croisor®100, developed by DuPont) in Europe, and colfencent (known as Genesis®, developed by Monsanto) in the USA, are the few reliable and registered gametocides. These gametocides are characterized by their reduced phytotoxicity and are applicable to genetically diverse parents (Curtis et al. 2002; Dennis 1998; Schachschneider 2018; Mcrae 1985). In China, a gametocide, SQ-1 (a Pyridazine compound), is being used for large-scale hybrid wheat production. SQ-1 has also demonstrated a low phytotoxic effect and can induce complete male sterility in maternal plants (Song et al. 2014; Wang et al. 2015, 2018). Since the 1980s, many hybrids in wheat have been developed and commercialized using CHAs. This approach has simplified hybrid development by eliminating the need for a maintainer line (Whitford et al. 2013). Likewise, it is found that CHA also plays a role in improving pre-available male sterility in a cytoplasmic male sterile (CMS) system (Titan et al. 2020). Despite their potential, CHAs are not yet considered an efficient system for large-scale hybrid seed production, primarily due to the incomplete induction of male sterility. The effectiveness of CHAs can be limited due to genetics of the genotype, composition of chemicals, and conditions of the environment. Determining the optimal dosage for a new parent line often requires extensive research. Further, the use of certain statistical methods can improve the efficacy of a CHA across diverse genotypes (Easterly et al. 2019). Table 2 incorporates a list of renowned commercially utilized CHAs for hybrid wheat development.

Table 2.

Renowned commercially utilized chemical hybridizing agent (CHAs) causing male sterility in wheat

CHA	Developer	References
Hybrex	Rohm & Haas	Mcrae (1985)
Crosior	DuPont	Schachschneider (2018)
Genesis	Monsanto	Dennis (1998)
SQ-1	China	Wang et al. (2015)

Cytoplasmic Male Sterility (CMS)

CMS Sources in Wheat

CMS is considered as most common source of male sterility in major crops including sorghum, rice, maize and wheat. Cytoplasmic male sterility (CMS) in wheat is caused by mutations in mitochondrial DNA or its interaction with nuclear and cytoplasmic genetic factors (Chase and Gabay-Laughnan 2004; Hanson 2004; Horn 2006). Mainly it is developed by inter or intraspecific hybridization between wheat wild relatives (Kaul 1988; Budar and Pelletier 2001; Melonek et al. 2021). CMS was first identified during 1951, when cytoplasm of Aegilops caudata was transferred to T. aestivum (Kihara 1951). Another successful source of CMS was discovered in Triticum timopheevii Zhuk (Wilson 1962a; b). Studies proved that mitochondrial signaling pathways and changes in the expression of nuclear genes are causing cytoplasmic male sterility in wheat (Chase 2007). Abnormality in fatty acid formation, lipid transport, polysaccharide metabolism and higher concentration of abscisic acid result in the pollen abortion in anthers of male sterile lines in wheat (Hao et al. 2021; Shokat et al. 2020, 2021a, b, 2024).

The cytoplasm from 35 species and wild relatives of Triticeae were tested in common wheat, with 20 found as a source of complete or partial male sterility (Adugna et al. 2004). Important sources of male sterility are T-CMS, K-CMS and V-CMS having sources from T. timopheevii, Aegilops kotschyi, and A. variabilis, respectively (Wilson 1962a, b; Mukai and Tsunewaki 1979). Another novel source of male sterility in wheat is derived from the cytoplasm of Hordeum chilense (wild barley) and is named as msH1. It was stable under different environments with complete and effective male sterility in a wide range of genotypes. It was also reported to be effective if there is a delay in flowering of the restorer line and some differences in plant height exist (Martín et al. 2008).

Triticum timopheevii is considered a major source of cytoplasmic male sterility with no undesired effects on grain yield and provides complete and stable male sterility under different environmental conditions (Mukai and Tsunewaki 1979; Adugna et al. 2004; Singh et al. 2010; Longin et al. 2012). Moreover, T. timopheevii is a favorable choice as compared to other sources of CMS because it has both recessive alleles which are the main contenders for male-sterility as well as male fertility restorer alleles (Wilson and Driscoll 1983). QTLs identified against T. timopheevii cytoplasm were located on chromosomes 2A, 4B and 6A (Ahmed et al. 2001). CMS system of T. timopheevii is being utilized for commercial hybrid seed production in different countries including India, Australia, USA, China and South Africa (Koekemoer et al. 2011; Longin et al. 2012). In cultivated wheat, CMS lines are developed commonly by crossing potential wheat genotypes with cytoplasmic male sterility source i.e., T. timopheevii or species of Aegilops and Hordeum. Then resulting F₁ is backcrossed with cultivated wheat for 5 to 6 generations (Wilson and Ross 1962a, b; Martín et al. 2008; Mukai and Tsunewaki 1979). Hybrid seed production through cytoplasmic male sterility is comprised of a 3-line system called A, B, and R-line. Here, the A-line is a female (male sterile), the B-line is genetically like the A-line but has a fertile cytoplasm also called as maintainer line. R-line is a male parent used for the restoration of fertility.

Fertility Restoration in CMS

A significant proportion of the fertility restoration genes (Rf) initially originated from Triticum timopheevii (Edwards 1983). Additional sources of male fertility in wheat were developed from T. spelta and the T. dicoccoides var. spontaneovillosum (Panayotov et al. 1986). The genotypes of T. timopheevii, T. aestivum, T. dicoccoides, T. spelta, H. chilense, and A. comosa (Table 3) have been utilized for the restoration of fertility in male sterile lines (Oehler and Ingold 1966; Panayotov et al. 1986; Martín et al. 2008). Wheat hybrids cultivated in various regions around the world reveal a varying degree of instability and environmental sensitivity linked to the fertility restoration genes within the CMS system (Jošt and Lucken 1983). In CMS wheat, complete fertility restoration in the F₁ generation requires the presence of two or three Rf genes, along with additional modifier genes (Bahl and Maan 1973; Du et al. 1991; Robertson and Curtis 1967; Lukaszewski 2017; Ma and Sorrells 1995). Fertility restoration in plants is a multifaceted trait influenced by different genetic effects, including monogenic, digenic, and polygenic factors (Sage 1976; Shahinnia et al. 2020). Male fertility restoration in wheat using R lines also requires the interaction of multiple genes. Two dominant and two additive genes were identified as responsible for facilitating male fertility restoration in wheat (Panayotov et al. 1986).

Table 3.

Different sources of restoration of cytoplasmic male fertility

Gene	Chromosome no	Fertility source	References
Rf1	1A	T. timopheevii × Marquis	Robertson and Curtis (1967)
Rf2	6B	[(T. timopheevi × A. squarrosa) × Canthatch]	Yen et al. (1969)
Rf3	1B	R18 & R9034	Zhou et al. (2005)
Rf4	7D	[(T. timopheevi × A. squarrosa) × Dirk]	(Yen et al. 1969)
Rf8	2DS	PWR4099	Sinha et al. (2013)
Rf9	6AS	Gerek 79	Shahinnia et al. (2020)
Rfm1	1BS	Chinese Spring	Tsujimoto (1984), TA et al. (2001)
Rfv1	1BS	Chinese Spring	Mukai and Tsunewaki (1979)
Rf6HchS	6HchS	H. chilense	Martín et al. (2008), Castillo et al. (2014)
Rf multi	1BS	Pavon 76	Tsunewaki (2015)
Rfd1	1BS	LK783	Niu et al. (2022)

The fertility restoration in wheat has been observed to be influenced by multiple mutant recessive genes as evidenced in experiments involving ethyl methane sulfonate (EMS) mutagenesis. These recessive genes exhibit mirror effects and significantly impact the fertility restoration in wheat (Murai et al. 1995). The presence of several modifier genes on chromosomes 1B, 4B, and 4D has been indicated other than major fertility restoration genes located on chromosomes 1A and 6B (Table 3). Furthermore, the inhibitors of male fertility restoration found on chromosomes 5A, 6A, and 5B have also been investigated (Maan et al. 1984; Geyer et al. 2018). The QTL analysis found Rf1 gene situated on chromosome 1A and its interaction with other modifier genes located on chromosome 1BS, as well as with another restorer locus, Rf4, on chromosome 6B. These modifiers significantly influence the expressivity and penetrance of the Rf1 gene, thereby playing a crucial role in determining its effectiveness in fertility restoration (Geyer et al. 2018). It is hypothesized that genes distributed across all 21 chromosomes in hexaploid wheat may contribute to the restoration of male fertility in wheat. This indicates a broad genomic involvement in the fertility restoration process (Du et al. 1991; Chen and Wehling 2003).

A male sterile line was created by incorporating the cytoplasm of H. chilense, resulted in the msH1 cytoplasmic male sterility (CMS) system. The fertility restoration line within this system developed by transferring the 6S chromosome from H. chilense accession H1 (6H^chS) into the hexaploid wheat'Chinese Spring' (Martín et al. 2008). Later, the identification of a second Rf gene on the 1H^chS chromosome supports the concept that two or more genes control fertility restoration in CMS systems (Castillo et al. 2014). Recent advancements in transcriptomic analysis and chromosome engineering have led to the mapping of six candidate genes on the 6H^chS chromosome within the msH1 system of male sterility (Rodríguez-Suárez et al. 2020).

In CMS, traditional hybrid seed production is based on the utilization of T. timopheevii but maintaining the three necessary lines in this system is a challenging and time-consuming process. It also requires more inputs compared to hybrids produced in cross-pollinating plants. Complete fertility restoration using restorer (R) genes often results in variable penetrance and expressivity (Liu et al. 2002; Geyer et al. 2018). The interactions between nuclear and cytoplasmic factors in plants are not yet fully understood, and this lack of clarity contributes to the complexity of male sterility and fertility traits. These traits can also exhibit unstable effects when subjected to different environmental conditions (Tsunewaki 1993; Ikeguchi et al. 1999).

Nuclear Genetic Male Sterility (NGMS)

Male sterility induced by mutations, transposon insertions, and regulatory disruptions in the nuclear content of a plant's genome, is known as nuclear, genic, or genetic male sterility (GMS). Compared to cytoplasmic male sterility (CMS), research on the application of genic male sterility (GMS) in plants has been relatively limited (Kaul 2012). However, GMS offers several advantages over CMS, including a broader selection of parental lines for hybrid development. Additionally, GMS allows avoidance of negative effects associated with the transfer of wild cytoplasm for induction of male sterility, compared to CMS (Rao et al. 1990; Kaul 1988).

Previous studies on wheat lines through spontaneous and induced mutations opened a new window to achieve male sterility through recessive genes such as ms1a, ms1b, and ms1c (Fossati and Ingold 1970; Driscoll 1977; Suneson 1962; Liu et al. 1996). The dominant mutant genes consist of the Ms2, Ms3 and Ms4, located on chromosome arm 4DS, 5AS and 5DS respectively, were found (Bing-Hua and Jing-Yang 1986; Maan et al. 1987; Qi and Gill 2001; Maan and Kianian 2001). Similarly, the recessive mutant genes ms1 and ms5, located on chromosome arm 4BS and 3AL, have also been identified (Klindworth et al. 2002). Seven allelic forms of ms1 locus were reported and named as ms1a, ms1b, ms1c, ms1d, ms1e, ms1f, and ms1g (Zhou and Wang 2007; Suneson 1962; Sasakuma et al. 1978; Zhou et al. 2008). A novel ms1s allele of ms1 gene has been mapped on the 4BS chromosome and can be used as genetic markers in selective breeding for NGMS (Yang et al. 2021a).

Driscoll in 1972, first proposed hybrid seed production through nuclear genic male sterility in wheat using XYZ system (Driscoll 1972, 1985). This innovative approach involved the development of three distinct lines of wheat, each characterized by the presence of a homozygous recessive mutant gene, ms, located on all three homeologs of chromosome 5 in the genome. A key aspect of this system is the incorporation of an additional alien chromosome, 5R, derived from rye (Secale cereale), which possesses a corresponding Ms gene that confers fertility and a phenotypic marker gene that results in a hairy peduncle (Driscoll 1985). The XYZ system comprises three lines: (i) The X-line is genetically male fertile, distinguished by its homozygous recessive ms state and the presence of two homologous from an alien chromosome. (ii) The Y-line is also genetically male fertile and homozygous recessive but contains a single homolog of the alien chromosome. (iii) The Z-line is homozygous recessive and genetically male sterile, lacking any dose of the alien chromosome. A crucial feature of this system is the non-pairing of the 5R rye chromosome with any of the wheat chromosomes, ensuring genetic stability across generations. Self-pollination of the X-line results in fertile offspring, while the Y-line produces two types of gametes, leading to offspring that are 75% male sterile (ms) without the alien chromosome and 25% male fertile, identical to the Y-line itself. This distribution allows for the use of the 75% male sterile (Z-type) plants as the female line in hybrid seed production, while the remaining 25% male fertile (Y-type) plants serve to maintain the Z-line through selfing. Despite its theoretical promise, Driscoll acknowledged potential challenges with the XYZ system, including the risk of contamination and the instability of male sterility and fertility in subsequent generations (Driscoll 1972). These concerns, coupled with the practical difficulty of selecting ms/ms plants based on the presence of a hairy peduncle in field conditions, led to the conclusion that the XYZ system could not be feasibly implemented in wheat hybrid seed production.

An enhanced version of the XYZ system has been identified and designated as the 4E-ms system (Zhou et al. 2006; Zhou and Wang 2007). Within this framework, seed color serves as a phenotypic marker to differentiate between male sterile and male fertile seeds. The phenomenon of blue grain, initially investigated in hexaploid wheat, emerged through wide hybridization efforts with the Agropyron spp. (Keppenne and Baenziger 2011). The blue aleurone trait was leveraged as a morphological marker within the 4E-ms system for the selection of male sterile lines. In this system (Fig. 2), a mutant ms line of hexaploid wheat (2n = 42 msms), was hybridized with a 4E disomic addition line (2n = 42 + 2E = 44, MSMS) derived from Agropyron elongatum ssp. Ruthenicum (Endo et al. 1991). The progeny from this cross in the F₁ generation, emerged as a monosomic 4E addition line (2n = 42 + 1E = 43, MSms), demonstrating male fertility, while its seeds exhibited a light blue grain color (Endo et al. 1991). This phenotypic characteristic is attributed to the alien chromosome 4E, which contains the Blue Aleurone (BA) gene in addition to a gene responsible for male fertility. Upon self-pollination, the F₁ generation yielded three distinct seed color phenotypes: deep blue, light blue, and white. These phenotypes correspond to the presence of two, one, and zero copies of the 4E chromosome, respectively, as delineated in Fig. 2. The study showed a seed color distribution of 64.3% white (male sterile), 32.1% light blue (monosomic male fertile) and 3.6% deep blue (disomic male fertile). These white aleurone seeds were deemed as Z-lines that can be used as male sterile lines for hybrid seed production, while the light blue aleurone seeds (male fertile lines) were deemed as Y-lines, they can reproduce the male sterile lines (Z-lines) again through self-pollination. This breeding scheme allowed any wheat cultivar to restorer fertility in F1 seeds upon crossing with the female, white-seeded male sterile line. While this system presents an improvement over the preceding XYZ system, its commercial applicability remains undocumented. The system raises concerns regarding genomic stability due to the presence of alien chromosomes and the complex linkage between the Ms1 and BA genes located on chromosome 4E, necessitating further investigation. Additionally, variability in the penetrance of ms1 mutations leads to concerns over the potential mixing of male sterility lines in F₁ hybrid progeny (Tucker et al. 2017). Moreover, environmental factors can also lead to variations in seed color, introducing bias in the development of male sterile lines within this system.

Fig. 2.

The 4E System of genic male sterility in hexaploid wheat. This 4E systems indicating that male sterile hexaploid wheat line becomes male fertile due to 4E disomic addition line of Agropyron elongatum. Further, F₁ generation produces three types of aleurone color seeds, of which light blue aleurone seeds were identical to F₁ and could be used again to maintain sterility. White aleurone seeds are male sterile and cand be utilized as a female parent in hybrid development

Molecular approaches and genetic engineering have been proposed for inducing male sterility through recessive genes such as ms5 and Tams45 (Pallotta et al. 2019; Singh et al. 2018). A notable method involves targeted mutations in the Ms45 gene across all three homeologs of hexaploid wheat using the CRISPR-Cas9 system. Furthermore, some related systems of hybrid seed production including seed production technology tested in other crops and a split-gene system, could be utilized as viable strategies in wheat for hybrid seed production (Wu et al. 2016; Kempe et al. 2014). These methods circumvent the issues associated with linkage breakage between fertility and marker genes in hybrid seed production. Additionally, dominant genes for male sterility, such as Ms1 and Ms2, have been identified through molecular techniques as a potential approach known as transgenic genetic male sterility and has been reported in several studies (Ni et al. 2017; Tucker et al. 2017).

Environment-Sensitive Male Sterility

Male sterility can also be achieved by manipulating environmental factors such as differences in temperature, photoperiod, and both temperature-photoperiod. It is referred as environmental sensitive male sterility. This type of male sterility is beneficial by utilizing a two-line hybrid system in which a maintainer line is not required as compared to the three-line system in CMS and GMS (Zhou et al. 2011; Mukai and Tsunewaki 1979). This system of male sterility in wheat provides advantages to identifying the range of best combiners, easy multiplication of male sterile lines, and less cost of hybrid seed production (Li et al. 2006). The detailed information is described below.

Photoperiod Sensitive Male Sterility

In photoperiod-sensitive male sterility, pollen transitions occur from viable to non-viable as the day length exceeds 14 h (Murai and Tsunewaki 1993; Murai 2001). This phenomenon negates the need for a maintainer line since male sterility is preserved through self-pollination (Mukai and Tsunewaki 1979; Sasakuma 1979; Murai et al. 2008). Photoperiod-sensitive cytoplasmic male sterility (PCMS) was introduced into the common wheat cultivar'Norin 26' through cytoplasmic substitution from Aegilops crassa, followed by selective breeding to ensure the expression of photoperiod-sensitive male sterility while maintaining agronomic viability (Murai and Tsunewaki 1993).'Norin 26' with substituted cytoplasm exhibits male sterility under long-day conditions—when the day length exceeds 15 h—by transforming its stamens into pistils during the floret differentiation stage. Conversely, it remains male fertile under short-day conditions with less than 14.5 h of day lengths. This dual response to photoperiod length has been thoroughly investigated in several studies (Mizumoto et al. 2011; Murai et al. 2002a, 2016).

PCMS in wheat is preserved under short-day conditions through self-pollination, enabling the production of hybrid seeds by crossing the male sterile line with a fertile male parent under long-day conditions exceeding 15 h. Fertility restoration within the PCMS framework is regulated by multiple genes (Rf), as shown in Norin 61. These genes have been localized to chromosomes 4A, 3D, 1D, and 5D (Murai 1997). Additionally, a single dominant gene, Rfd1, identified in the'Chinese Spring' wheat cultivar and situated on the 7BL chromosome, along with 20 modifier genes, has also been implicated in the control of fertility restoration in the PCMS system (Murai and Tsunewaki 1994; Murai et al. 2002a).

Another concept of photoperiod-mediated male sterility known as photoperiod genic male sterility (PGMS), initially successful in rice, has been explored in wheat to facilitate hybrid seed production (Shi 1985). Furthermore, a unique form of male sterility, sensitive to both low temperature and short-day conditions, was identified in the'Gaining 14' cultivar in Hunan, China (He 1993).

Thermosensitive Male Sterility

Thermo-sensitive genic male sterility (TGMS) in wheat was first identified in the 1970s (Jan 1974). Research has shown that wheat is sensitive to both low temperatures (4 °C) and high temperatures (25 °C) during meiosis (Tepliakov et al. 1974; Campbell and Davidson 1979). Notably, abnormal cytokinesis in the thermo-sensitive wheat line'B366' at a low temperature of 10 °C has been attributed to the suppression of cytokinesis-associated genes. This suppression leads to male sterility, thus enabling the development of hybrid wheat varieties (Tang et al. 2011; Liu et al. 2021). Another TGMS wheat line,'BNY-S', has been identified as a spontaneous mutation originating from the fertile wheat line'BNY-F'. The sterility of'BNY-F' is controlled by a single recessive gene, wtms1, located on chromosome 2B. This gene renders the plant male sterile at temperatures below 10 °C during the spikelet differentiation stage, whereas it remains male fertile at temperatures above 10 °C (Xing et al. 2003). Another TGMS line controlled by a recessive gene, tmsBS20T, located on chromosome 2BL, results in complete male sterility at temperatures below 10 °C and ensures full male fertility at temperatures above 13 °C. Such temperature-dependent fertility has been instrumental for hybrid seed production in China (Ru et al. 2015). A study elucidated the role of microRNAs (miRNAs) and trans-acting small interfering RNAs (ta-siRNAs) in the induction of TGMS, by regulating the auxin signaling pathways at low temperatures (Tang et al. 2012).

Similarly, high temperatures have been associated with TGMS in wheat, attributed to a reduction in auxin levels in specific tissues. The application of supplementary auxin hormones at high temperatures has been shown to induce male fertility in wheat (Sakata et al. 2010). The TGMS line'4110S' has been reported as male sterile at temperatures exceeding 20 °C, yet it remains fertile under cooler conditions (Yang et al. 2018). A recent study has identified two genes, TaMut11 and TaSF3, in wheat that are crucial in pollen development and mutations in these genes facilitated the development of TGMS under high-temperature conditions (Yang et al. 2021b). Another study identified that the silencing of these genes resulted in a marked decrease in fertility, thus providing new insights into the underlying mechanisms of wheat pollen development within the context of TGMS (Hao et al. 2021).

The male sterility gene in the YM-type of TGMS wheat line has been identified on chromosome 1BS (Puhui et al. 2006). Chromosomal mapping has revealed a TGMS gene, tmsBS20T, on chromosome 2BL in the'BS20-T' hexaploid wheat line (Ru et al. 2015). The'BS366' line, utilized as the female parent in developing numerous hybrid wheat varieties in China, becomes male sterile at 10 ℃ with 12–14 h day length and becomes male fertile at 20 ℃ with 12–14 h day length. Genome-Wide Association Study (GWAS) investigating DNA methyltransferase (DMT) genes in BS366 wheat line identified 52 DMT genes—a count significantly surpassing those found in Arabidopsis and rice. This study also conducted a correlation analysis, pinpointing three DMT genes—TaCMT-D2, TaMET1-B1, and TaDRM-U6—that might be involved in male sterility. These genes can be considered as candidate genes contributing to TGMS in the'BS366' wheat line (Yuan et al. 2020).

Thermo-sensitive cytoplasmic male sterility (TCMS) has also been explored through the use of Aegilops kotschyi cytoplasm and a male sterile gene from Triticum spelta, resulting in the designation of this system as the YS type of TCMS (He 2000). Within this YS-TCMS framework, Ae. kotschyi cytoplasm and the 1BS chromosome from Triticum spelta was incorporated in the common wheat line'KTP3314A'. This line exhibited male sterility at temperatures below 18 °C for 3–9 days preceding the heading stage and displayed partial male fertility at temperatures above 20 °C (Song et al. 2013; Meng et al. 2016). Another TCMS line'FA99' was identified as male sterile at a temperature threshold of 12–14 °C due to pollen abortion at the pollen mother cell formation stage. In this study, transcriptome analysis studied 252 differently expressed genes involved in multiple biochemical pathways related to pollen development in the'FA99' TCMS line (Liu et al. 2022). TaEXPB5, a thermo-sensitive cytoplasmic male fertility-controlling gene was identified in the'KTM3315A' wheat line and it up-regulated the fertile anthers. Moreover, the silencing of this gene eliminates the effects of temperature on controlling male fertility, depicting its major role in the TCMS system in wheat (Geng et al. 2021).

Photo-Thermo-Sensitive Male Sterility

Another system known as photo-thermo sensitive genic male sterility (PTGMS), in which a line'BS210', becomes male sterile at 10–12 ℃ with 10–12.5 h day length as well as male fertile at 8–10 ℃ and 14 h day length during the anther development stage (Sun et al. 2017). Another PTGMS wheat cultivar'337S', three thermos-photo-period sensitive male sterility controlling genes, wptms1, wptms2, and wptms3, have been mapped to chromosomes 5B, 2B, and 1BS, respectively (Guo et al. 2006; Chen et al. 2011). Furthermore, three quantitative trait loci (QTLs) associated with PTGMS were identified in the wheat cultivar'BS366' on chromosomes 2DS, 4BS, and 7AL, named QF.bhw-2DS, QF.bhw-4BS, and QF.bhw-7Al, respectively (Yuan et al. 2020).

All systems of environmentally controlled male sterility including PGMS, PCMS, TGMS, TCMS, and PTGMS, share a common limitation: their suitability for specific geographical regions (Virmani 2003). Additionally, in the environmentally controlled two-line male sterility system, the failure to achieve complete male sterility and challenges in fertility restoration due to sudden geographic effects present significant obstacles (Dai et al. 2008).

All conventional approaches discussed above for hybrid wheat breeding are labor-intensive and different from normal wheat breeding (summarized in Table 4 and Fig. 3). These approaches also indicate several limitations in hybrid seed production, including but not limited to the inability to achieve complete sterility, restoration of complete fertility in the first filial generation (F₁), and concerns related to the transfer of cytoplasmic elements during CMS development. To address these limitations, molecular approaches should be explored to discover new potential to utilize in hybrid wheat production.

Table 4.

Different features of normal wheat breeding and hybrid wheat breeding

Features	Normal wheat breeding	Hybrid wheat breeding
Pollination	Self or cross-pollination	Cross pollination
Male sterility	Not required	Essential
Parent types	True breeding lines	Male sterile × Restore lines
Genetic variation	Low (Homozygous)	High (Heterozygous)
Heterosis (Vigor)	Low	High
Seed production	Require fewer resources	Require more resources

Fig. 3.

This figure showing different techniques utilized to develop male sterility in wheat for Hybrid Development. A Emasculation: Involves removing anthers from wheat florets using scissors and forceps or hot water treatment. B Chemical Hybridizing Agents (CHAs): Male sterility induced by the application of certain chemicals before anthesis. C Cytoplasmic male sterility (CMS): Male sterility is induced by mutations in mitochondrial DNA or its interaction with nuclear and cytoplasmic genetic factors. It includes the utilization of a 3-lines system in which the (B) line is used for the maintenance of sterility, and the male sterile (A) line is crossed with the restorer (R) line for the restoration of fertility to develop hybrid wheat. D Genic Male Sterility (GMS): Male sterility induced by mutations, transposon insertions, and regulatory disruptions in the nuclear content of a plant's genome. It involved the utilization of the 2-line system (A) and (R) line for Hybrid development. E Environmental Male Sterility (EMS): Male sterility in the female line is induced by manipulating the environmental factors, such as differences in temperature, photoperiod, or both temperature and photoperiod. F Gene Editing: Induction of male sterility by employing the modern molecular biology techniques, which include the precise mutation in the targeted gene related to male sterility and fertility. G Genetic Engineering: Inducing male sterility in the wheat lines by incorporating a foreign genetic source from a biological identity

Molecular Approaches for Hybrid Seed Production in Hexaploid Wheat

Biotechnology has introduced numerous unique ways of causing male sterility, which refers to the creation of male sterile plants that can be turned on and off by external stimuli like light, temperature, or chemicals (Perez-Prat and van Lookeren Campagne 2002). Advanced biotechnological approaches offer promising avenues for addressing the inherent challenges in traditional hybridization methods. Marker-assisted selection, i.e., the use of molecular markers to identify and select desirable traits in breeding populations, represents one of the earlier molecular tools that improved breeding precision (Collard and Mackill 2008). Transcriptional gene silencing, i.e., the targeted suppression of gene expression at the RNA level, introduced a more refined control over specific genes (El-Sappah et al. 2021). The development of transgenic plants with introduced novel traits, i.e., inserting foreign genes to confer new characteristics such as pest resistance or stress tolerance, marked a significant leap in crop improvement (Anwar and Kim 2020). Modifying the naturally enclosed structure of wheat florets to a partially open structure through molecular approaches, i.e., altering floral architecture to enhance cross-pollination, enables increased hybrid seed development (Whitford et al. 2013). Most recently, the exploration of split-gene systems, i.e., genetic constructs designed to activate gene expression only under specific conditions by separating essential gene components, provides an advanced level of regulatory control (Kempe et al. 2014). These technologies contribute to more efficient, accurate, and sustainable hybrid seed production in hexaploid wheat.

Marker Development and Marker-Assisted Selection

Hybrid seed production in common wheat can be improved by marker-assisted selection (MAS). Historically, morphological markers such as aleurone color and hairy peduncle have been utilized for the selection of male sterile and fertile lines (Driscoll 1972; Keppenne and Baenziger 2011). However, these phenotypic markers are susceptible to environmental variation, leading to potential instability in the results. In contrast, molecular markers enable the selection of male fertile and sterile lines at the genotypic level independent of environmental variations will be more useful. The bulked segregant analysis alongside high-throughput genotyping techniques emerges as a potent method for identifying QTLs, alleles, and markers associated with various phenotypic traits, such as male sterility, male fertility, and essential floral traits for hybrid seed development in hexaploid wheat (Zou et al. 2016; Cao et al. 2009). Historically, markers linked with male sterility are summarized in Table 5.

Table 5.

Molecular markers identified for hybrid seed production in wheat

Marker name	Marker type	Associated gene/locus	Functionality	Chromosome position	Specie name	References
MS2-WMC617	SSR	Ms2	Male sterility	4DS	T. aestivum	Cao et al. (2009)
S5A_138430506 S5A_266997650	SNP	Ms3	Male sterility	5A	T. aestivum	Guttieri (2020)
ms1sCAPS1 ms1sCAPS2 ms1sCAPS3	CAPS	ms1s	Male sterility	4BS	T. aestivum	Yang et al. (2021a)
Xgwm 374	SSR	Wtms1	Male sterility	2B	T. aestivum	Xing et al. (2003)
Xgwm335	SSR	wptms1	Male sterility	5B	T. aestivum	Guo et al. (2006)
Xgwm374	SSR	Wptms2	Male sterility	2B	T. aestivum	Guo et al. (2006)
Xgwm182	SSR	wptms3	Male sterility	1BS	T. aestivum	Chen et al. (2011)
Xgwm18 Xwmc406	SSR	rfv1-1(Locus)	Male sterility	1BS	T. aestivum	Ju-Hong et al. (2010)
gwm403 gwm374	SSR	tmsBS20T	Male sterility	2BL	T. aestivum	Ru et al. (2015)
Xbarc95 Xgdm35	SSR	taf1(Locus)	Female sterility	2DS	T. aestivum	Dou et al. (2009)
AX-94501544 AX-94682405	SNP	Rf1	Male fertility	1A	T. timopheevii	Geyer et al. (2018)
Xbarc207 Xgwm131 Xbarc61	SSR	Rf3	Male fertility	1B	T. timopheevii	Zhou et al. (2005)
IWB72107	SNP	Rf3	Male fertility	1BS	T. aestivum	Geyer et al. (2016)
Xbcd249 Xcdo442	RFLP	Rf3	Male fertility	1BS	T. aestivum	Ma and Sorrells (1995)
Xksug48	RFLP	Rf4	Male fertility	6B	T. aestivum	Ma and Sorrells (1995)
Xwmc503	SSR	Rf8	Male fertility	2DS	T. aestivum	Sinha et al. (2013)
IWB72413	SNP	Rf9	Male fertility	6AS	T. aestivum	Shahinnia et al. (2020)
Xnwafu1	SNP	Rfd1(Locus)	Male fertility	1BS	T. aestivum	Niu et al. (2022)

Marker-assisted selection (MAS) can be beneficial in the early stages of hybrid development and can differentiate the germplasm by male sterility and fertility genes. However, its application at a commercial level or in large-scale hybrid seed production is still not much popular.

Genetic Engineering Systems for Male Sterility in Hybrid Wheat

The first genetically modified system for hybrid seed production, centered on a fertility control mechanism, was tested in 1997 (Block et al. 1997). In this innovative approach, the Barnase gene, sourced from the bacterium Bacillus amyloliquefaciens, was introduced into hexaploid wheat using the biolistic transformation method. The gene expression was driven by tapetum-specific promoters derived from rice and corn. This strategic expression led to the early-stage degradation of the tapetum layer, an essential nutrient source for pollen development, thereby inducing sterility in wheat spikelets. Notably, in this system, glufosinate resistance was integrated alongside the sterility gene through Bayer’s SeedLink® technology. This integration facilitated the early field-level identification and selection of male sterile lines (Block et al. 1997). The Split-Transgene System, a more recent development, segregates the Barnase gene into two distinct loci situated on separate chromosomes. Upon simultaneous expression, these separated gene fragments synthesize a functional cytotoxin. This innovation facilitates the straightforward maintenance of male-sterile female parent lines and the subsequent restoration of male fertility in the first-generation (F₁) hybrids (Kempe et al. 2013, 2014).

Genome Editing Systems for Exploiting Male Sterility

Modern genome editing technologies, particularly CRISPR/Cas9, have revolutionized our ability to induce male sterility in wheat by offering precise and efficient methods for creating male-sterile lines which are crucial for hybrid breeding. This section reviews recent advances in applying these biotechnological approaches to exploit male sterility in wheat.

CRISPR/Cas9 has emerged as a powerful tool for inducing male sterility in wheat by targeting specific genes involved in pollen development. This approach is considered precise and reliable for breaking breeding obstacles in wheat (Farinati et al. 2023). Okada et al. (2019) reported a significant breakthrough in inducing nuclear male sterility in wheat using CRISPR/Cas9. By precisely targeting and knocking out the Ms1 gene, they generated four T2 knockout mutants that appeared to be non-transgenic. This development offers great potential for creating commercially viable hybrid wheat varieties (Okada et al. 2019). Further, PhasiRNAs are important regulators of anther development in plants and are essential in plant male sterility (Fan et al. 2016). A recent study by Niu et al. (2022) focused on genes crucial for phasiRNA biogenesis in wheat, including TaDCL4, TaDCL5, and TaRDR6. Their findings revealed that CRISPR/Cas9-based mutation of the TaDCL5 gene resulted in normal, healthy wheat plants that exhibited male sterility, with no reported difference in pollen grain number compared to the wild-type.

A critical player in gametophyte development in wheat is the gene TaNP1 (Almutairi 2022), which is an ortholog of OsNP1 in rice and ZmIPE1 in maize (Zhenyi et al. 2016), as playing a key role in male gametophyte development. CRISPR/Cas9-mediated targeted mutations in this gene resulted in complete male sterility. Research into cytoplasmic male sterility (CMS) coupled with nuclear Restorer-of-fertility (Rf) genes has provided crucial insights into fertility restoration mechanisms (Chase 2007). Melonek et al. (2021) found that Rf1 and Rf3 genes bind to the mitochondrial orf279 transcript and induce cleavage, preventing expression of the CMS trait. The identification of these genes and orf279 as the genetic determinant of T-CMS represents a significant breakthrough for enhancing fertility restoration and maximizing yield gains from F₁ hybrids. Singh et al. (2022) and Whitford et al. (2013) demonstrated that transcriptional gene silencing in hexaploid wheat effectively silenced three anther-specific genes i.e., TaMs45, TaMs1, and TaEXPB5, resulted to male sterility. Okada et al. (2018) described a technique involving constitutive RNA expression of a promoter inverted repeat (pIR), which suppressed the transcription of target genes. This approach also proved effective for silencing male fertility genes across the three wheat sub-genomes, overcoming the challenge of functional redundancy in hexaploid wheat.

Hybrid Wheat Production Status Around the Globe

Hybrid wheat breeding efforts have predominantly taken place in countries with extensive wheat cultivation areas. France, South Africa, Australia, and the USA are known for commercial hybrid wheat production. Additionally, numerous countries, including Pakistan, China, India, the UK, the former USSR, the Netherlands, Yugoslavia, Mexico, Japan, Italy, Hungary, Canada, Bulgaria, Germany, Belgium, and Denmark, are actively engaged in research and development programs for hybrid wheat (Braun et al. 2001; Longin et al. 2012). Notably, the USA initiated hybrid wheat production planning in 1957 and released the first commercial CMS-based hybrid in 1974 (Parodi and Gaju 2009). In Mexico, Europe, and Australia, both commercial and public sector projects have been initiated to support hybrid wheat breeding programs (Longin et al. 2013). In the USA, the development of hybrid wheat is pursued by private sector companies such as Syngenta, BASF, and DuPont-Pioneer, alongside collaborative efforts in the public sector, including wheat breeders from the University of Nebraska-Lincoln and Texas A&M AgriLife Research (Ledbetter 2016).

In 1982, hybrid wheat varieties were introduced in Europe and the USA, utilizing CMS and CHA Genesis by Monsanto. Since the 1990s, over 60 hybrid wheat varieties have been marketed in both regions (Parodi and Gaju 2009). For a period, H-007 was used for commercial hybrid seed production in Europe and the USA, and CHA WL84811 was utilized across the USA, Europe, China, South Africa, New Zealand, and Australia. However, their use was phased out due to limited applicability and adverse toxic residual effects, respectively. Until 2007, two innovative CHAs, Genesis™ and Croisor® 100 were extensively adopted in Europe and the USA. Presently, Genesis™ remains the only CHA commercially applied in the USA, while in Europe, hybrid production predominantly relies on CHA-based methods, with Croisor® 100 being the most commonly used (Boeven et al. 2016a; El Hanafi et al. 2022; Zhang et al. 2009; Wang et al. 2018). In 1994, the hybrid'Domino' was launched by HyberiTech-Monsanto in the USA,'Hpo-Precia' by Hybrinova in France, and'Hybnos1' by Nordsaat Saatzucht GmbH in Germany, marking their first hybrid release (Gupta et al. 2019).

In China, hybrid wheat research began in the 1980s, focusing on CHAs and CMS. To date, over 50 wheat hybrids have been trialed, with seven receiving approvals through provincial and national yield trials. This progress led to the formation of a hybrid wheat company in 2011 by China Seed at Beijing Academy of Agriculture and Forestry Sciences (BAAFS). In 2012, BAAFS developed two wheat hybrids that also underwent testing in Pakistan (Iftikhar Ali 2023). Currently, in Pakistan, hybrid seed production trials using the 4E system of genic male sterility (Blue Aleurone Technology) are under evaluation through the combined efforts of Pakistan’s research institutes and University of Sydney, Australia under the ACIAR project CROP 2020-167 (MNSUAM 2023; ACIAR 2024). Chinese wheat breeders have identified multiple variants of photoperiod-thermal sensitive male sterility (PGTMS) and thermo-sensitive genic male sterility (TGMS) in Yan Zhan 4110S, BNY-S, tmsBS20T, BNS, TGMS lines, BS366, and C49S PTGMS lines (El Hanafi et al. 2022; Zhang et al. 2001, 2003; Yang et al. 2018, 2021b; Xing et al. 2003; Li et al. 2009; Ru et al. 2015; Song et al. 2015; Liu et al. 2018). The hybrid wheat development program in India is based on CHAs and CMS systems. In 2002, Mahyco, a private sector company in India, launched its hybrid wheat production project and remained the sole producer of hybrid wheat in India. It successfully released two wheat hybrids using the CMS system (Matuschke et al. 2007). In 2009, the Indian Council of Agricultural Research (ICAR) launched a network project aimed to produce hybrid wheat through the CMS system, however, no significant results were reported (El Hanafi et al. 2022).

Likewise, the International Maize and Wheat Improvement Center (CIMMYT) also initiated a program using a CMS system derived from Triticum timopheevii, which was prematurely discontinued. However, research efforts resumed in the 1990s when CIMMYT collaborated with Monsanto, focusing on the development of a hybrid wheat production system in Northern Mexico using a CHA named Genesis. In 2011, CIMMYT started to collaborate with Syngenta to explore heterosis in CIMMYT's spring wheat germplasm. This partnership led to the evaluation of over 5,000 hybrids based on the CHA system in Mexico and India (Gupta et al. 2019; Murai and Tsunewaki 1993).

Conclusion and Future Prospects

The review dived into the current challenges and their potential solutions for wheat hybrid breeding that encompass to floral architecture, refining male sterility systems and utilization of conventional and molecular techniques. Heterosis in higher ploidy crop species is difficult due to the complex nature of the genome and scanty information about male sterility mechanism. We emphasized that wide hybridization could help to create lines with open floral architecture and to exploit hybrid vigor. But lack of efficient male sterility and fertility restoration systems poses challenges. Among available male sterility systems, Triticum timopheevii stands out to be an optimal choice to improve the cytoplasmic male sterility systems further due to its genetic advantage, adaptability, historical success, and widespread utilization. Therefore, CMS using Triticum timopheevii can be a better choice, but stable fertility restoration and inadequate understanding of cytoplasm-nuclear interactions are still the important challenges. Furthermore, the GMS system utilizing the blue aleurone (BA) gene offers a direct and feasible marker-based approach to identify the male sterility, which can significantly enhance the reliability and efficiency of hybrid breeding. Moreover, all the methods of environment-sensitive male sterility are found to be specific for a particular geographical location, which limits their utility on a broader scale. Modern trends in hybrid wheat breeding are expected to focus on developing innovative and stable male sterility and fertility systems using conventional and molecular approaches that can also reduce the costs associated with hybrid seed production (Lusser et al. 2012; Waltz 2012). Artificial nucleases, like CRISPR-based technologies, are designed to create site-specific DNA breaks, which the cell repairs, leading to targeted gene inactivation, gene addition, or trait stacking (Curtin et al. 2012; Lusser et al. 2012; Zhang et al. 2019). Conventional introgression and gene editing approaches can further be strengthened by incorporating modern cytogenomics techniques. Cytogenomics, an integrated approach of cytology and genomics has been widely utilized to visualize the behavior of alien introgressions (Orlovskaya et al. 2016; Silkova et al. 2018; Zhang et al. 2021), can play an efficient role in male sterility and fertility restoration (Badaeva et al. 2006). Cytogenomics could help to characterize those unnecessary cytoplasmic factors incorporated by cytoplasm addition and risk of cellular instability can be minimized (Lough et al. 2015). Modern cytogenomic approaches like fluorescent in situ hybridization (FISH) and genomic in situ hybridization (GISH) can precisely pinpoint the additional/substitutional chromosome or their segments carrying male sterility and fertility-inducing genes in GMS (Fukuhara et al. 2016; Zhang et al. 2021; Kosmala et al. 2006). Further, it can be utilized for precise mapping and screening of targeted homeologs and off-targets, respectively, before and after performing knockouts using modern genome editing tools to develop male sterility (Dhar and Koul 2024; Potlapalli et al. 2023). Targeted genome editing and simultaneous chromosomal tracking are now possible using modern tools in combination, such as CRISPR-Cas and FISH (Okada et al. 2019; Ishii et al. 2019; Wang et al. 2019). In genetic engineering, cytogenomic (FISH and GISH) could provide monitoring of transgenic stability in the targeted genome for developing male sterility and its maintenance in the next generations (Lukaszewski 2017). Furthermore, comparative genomics and transcriptomics technologies like whole genome sequencing (WGS) and RNA-Sequencing (RNA-Seq) are the other available options that could make it easier to study and characterize wheat male sterility and fertility-related genes through alien introgression, gene editing, and genetic engineering (Wang et al. 2017; Melonek and Small 2022; Bai et al. 2017; Ye et al. 2017; Rodríguez-Suárez et al. 2020). Thus, the integration of cytogenomics and comparative genomics along with conventional introgression approaches and related gene editing techniques holds immense potential to revolutionize hybrid wheat breeding by enabling the precise, efficient, and stable development of male sterility and fertility systems in future.

Abbreviations

CHAs

Chemical hybridizing agents

CMS

Cytoplasmic male sterility

GMS

Genetic male sterility

Rf

Fertility restoration

EMS

Ethyl methane sulfonate

ms

Male sterile

PCMS

Photoperiod-sensitive cytoplasmic male sterility

PGMS

Photoperiod genic male sterility

TGMS

Thermo-sensitive genic male sterility

TCMS

Thermo-sensitive cytoplasmic male sterility

PTGMS

Photo-thermo sensitive genic male sterility

QTLs

Quantitative trait loci

MAS

Marker-assisted selection

pIR

Promoter inverted repeat

FISH

Fluorescence In Situ Hybridization

GISH

Genomic In Situ Hybridization

WGS

Whole Genome Sequencing

RNA_Seq

RNA-Sewuencing

CIMMYT

International Maize and Wheat Improvement Center

Author Contributions

M.A. planned and conceptualized the review structure, wrote a part of the manuscript, U.S. contributed to writing a second major part of the manuscript, assisted in editing, and improved language for clarity. B.N. and S.H. both contributed to writing the sections of the manuscript. A.T.A. further refined the manuscript by improving its flow and conciseness. S.S. played a significant role in guiding, extensive editing, refining, and improving the language clutter to enhance the quality of the manuscript, as well as a communication author. All authors has proved the final version of the manuscript.

Funding

This work is not supported by any funding.

Data Availability

No datasets were generated or analysed during the current study.

Declarations

Competing Interests

The authors declare no competing interests.