Four different models have been described to explain how CMS can produce male sterility Chen and Liu (2014):

i) Cytotoxicity model: the proteins encoded by the CMS genes directly cause the death of the cells involved. At the basis of the mechanisms by which this occurs it has been hypothesized that there is mitochondrial dysfunction, but a well-defined model has not yet been developed, lacking molecular evidence of cytotoxicity itself. As a result, a simple explanation for CMS in these systems is that the CMS proteins cause mitochondrial malfunction in the anthers’ sporophytic or gametophytic cells, resulting in male abortion (Levings, 1993).

ii) Lack of energy model: the cellular respiration process is altered. In fact, CMS proteins can act as dysfunctional homologues of parts of complexes forming the electron transport chain, or changing proton gradients critical to the cellular respiration process, resulting in no ATP production. The molecular evidence supports the concept that some CMS are caused by an energy deficit in growing anthers, which demand more energy (Sabar et al., 2003; Wang et al., 2013).

iii) Asynchronous programmed cell death (PCD) model: PCD is induced in tapetum cells earlier than its normal course. It is implemented through the release of cytochrome C, a protein complex of the electron transport chain, and by increasing the production of reactive oxygen compounds (ROS, from Reacting Oxygen Species). By starting the autolysis before the pollen is mature, the tapetum cannot continue to nourish it and this does not complete its development. Plant male gametophytes form in anthers through cooperative contacts between sporophytic (anther wall) and gametophytic (microspore) cells, as well as correct PCD-controlled cellular degeneration of the tapetum, the deepest cell layer of the anther wall tissue (Mah, 2005).

iv) Retrograde regulation model: some CMS proteins are able to regulate the expression of nuclear genes, including some involved in the processes for correct reproduction. For example, they can disturb the formation activity of the stamens, in place of which carpels or petals develop. Or they can nullify the action of fertility restorative genes, when they are in their recessive allelic/haplotype form (Linke et al., 2003).

By comparing the proteomes of CMS and fertile lines, some CMS causative proteins, such as URF13 of maize CMS-T (Forde et al., 1978) and truncated COX2 of sugar beet CMS-G (Ducos et al., 2001), were discovered in other CMS systems. The CMS candidate genes were found in a few cases, such as radish CMS-Ogu (Bonhomme et al., 1991) and wheat alloplasmic CMS-AP (Rathburn and Hedgcoth, 1991), by analyzing the mitochondrial DNAs of segregating somatic hybrids (cybrids) produced from protoplast fusion between CMS-carrying lines and normal fertile lines. However, owing of the difficulties in acquiring cybrids and the uncertainty of recombination events between the mitochondrial DNAs of the fusion lines, this strategy is ineffective for most crops. Several methodologies can be used to identify CMS candidate genes. The most common approach is to look for changes in mitochondrial gene organization and/or mitochondrial transcriptome or proteome differences in CMS cytoplasm lines with and without the Rf gene(s). Nuclear Rf genes perform their action at different levels, involving various steps of protein synthesis or cellular metabolism (Chen and Liu, 2014).

Since in the case of CMS, 100% of offspring individuals will be MS, the use of molecular markers results a strategic key if identified and mapped in association with Rf genes. Table 1 shows main examples of the important crop classes for which mapped and retrievable information regarding reproducible, codominant molecular markers linked to Rf genes, offering fast and reliable detection tools to select, by MAS, parental lines for production of the desired progeny (Jordan et al., 2010; Yan et al., 2017).

In addition to major cereal crops and species, a great interest in mapping CMS-related loci has also been reflected in ornamental plants such as sunflower and petunia (Bentolila et al., 1998; Gentzbittel et al., 1999; Feng and Jan, 2008; Yue et al., 2010; Liu et al., 2012; Qi et al., 2012; Liu et al., 2013; Talukder et al., 2019).

Several molecular mechanisms underlie GMS in different species, in many of them genes coding transcription factors are capable of modifying the expression of genes involved in reproductive processes. The result is disturbance of gamete formation, due to failure of homologous chromosome separation in meiotic anaphase I and delayed of programmed cell death in tapetum (Jeong et al., 2014). In particular, several nuclear genes have been found responsible for MS, causing arrest of microspore development. Furthermore, as mentioned in Introduction section, the male sterility can also depend on environmental conditions, and in this case the GMS is define as EGMS. Temperature-sensitive genic male sterile (TGMS) and photoperiod-sensitive genic male sterile (PGMS) lines were developed especially in cereals crops like rice and wheat. TGMS lines are sterile at high temperatures and fertile at low temperatures, while PGMS lines can either be sterile when the day is longer than the night and fertile when it is shorter, or vice versa. In China, EGMS lines occupy 20% of the area dedicated to the cultivation of hybrid rice (Li et al., 2007). Also in this case, the molecular markers result strategic keys if identified and mapped in association with the ms locus, as testified in past (Barcaccia et al., 2016). However, to date, in more than 610 species of flowering plants the MS trait is under investigation, and specifically in the past few decades, at least 40 GMS genes have been identified by MAS and characterized in model Arabidopsis and rice (Chen and Liu, 2014; Singh et al., 2019; Wan et al., 2019). As similarly reported in Tables 1 , 2 shows main examples of the several crop classes for which mapped and retrievable information regarding reproducible, codominant molecular markers linked to nuclear male sterility genes. Molecular markers such as SSR, RFLP, SCAR, and SNP were fully employed for mapping male sterility genes, while insertion−deletion (InDel), target region amplification polymorphism (TRAP), sequence-related amplified polymorphism (SRAP), high resolution melting (HRM), and conserved orthologous set (COS) markers were sporadically used among these research studies. However, the data availability of mapped genes was correlated with species of great agronomic and economic importance: studies on cereals and other horticulture crop were prevalent, resulting in numerous mapped markers and associated genes involved in both MS systems, which offer open access for hybrid production using male-sterile lines (Li et al., 2007; Rout et al., 2021; Morales et al., 2022). These insights were interesting because proper MAS application could offer competitive phenotypes for market demand and contribute to reducing production costs, which is also fundamental for ornamental plant companies.

The CRISPR/Cas system, especially based on Cas9, has been successfully applied for the generation of male sterile lines in important worldwide food crops (Barman et al., 2019; Okada et al., 2019). Studies in main crops, such as rice, soybean, maize, and tomato, have reported that pooled CRISPR/Cas9 methods can result in valid strategies to generate a population of mutants for the MS trait (Jacobs et al., 2017; Meng et al., 2017; Liu et al., 2019; Liu et al., 2020). The application of CRISPR/Cas technologies for generating mutants with a male sterile phenotype is an effective tool, mainly mediating a knock-out approach towards target GMS genes with nuclear origin since, compared to cytoplasmic male sterile lines, it is much easier and more useful to produce hybrid seeds (Qi et al., 2020).

Generally, MS mutants result from mutations in target genes involved in microsporogenesis and/or microgametogenesis. Meiosis-related, tapetum-specific and transcription regulatory genes, such as eme1/exs, tpd1, ams and ms1, have been elucidated as key candidate genes involved in these biological processes (Canales et al., 2002; Zhao et al., 2002; Yang et al., 2003). Furthermore, many Arabidopsis transcription factors (TFs) genes, such as MYB103, DYT1, TDF1, AMS, bHLH10, bHLH89 and bHLH91, have been investigated as direct controllers of pollen development (Sorensen et al., 2003; Zhang et al., 2006; Zhang et al., 2007; Zhu et al., 2008; Zhu et al., 2015; Pan et al., 2020). The molecular and functional information has been then easily transferred from models to crops, as reported for Arabidopsis and rice, in which two analogous pathways regulating pollen and tapetum development have been identified in previous research (Fu et al., 2014; Jeong et al., 2014; Zhu et al., 2015; Mishra et al., 2018). In tomato, two homologous genes have been identified as regulators of tapetum and pollen formation. The first, SlMS10 (Solyc02g079810_ms10³⁵ ) gene, encoding a basic helix-loop-helix TF (bHLH) and homologue to AtDYT1 and OsUDT1, carries both PCD and meiosis alteration in the tapetum during microsporogenesis (Jeong et al., 2014). Its editing has confirmed that SlMS10 is a possible good target candidate for male sterility induction since its knockout mediated by the CRISPR/Cas9 system conferred a male sterility phenotype (Jung et al., 2020). Recently, Liu and colleagues demonstrated that the creation of a mutation in ms10³⁵ by CRISPR/Cas9 technology in association with its linkage marker genes led to marker use for creating mutants exhibiting complete male sterility and recognition during the early developmental stage, confirming promising application possibilities in the production of hybrid seeds (Liu et al., 2021a). The second, Solyc01g081100, homologous to the AtbHLH10/89/90 and OsEAT1 genes, is a candidate gene for the male sterile 32 (ms32) mutant, a locus affecting tapetum and pollen development, and for this reason, it is suggested as a good target for gene editing to quickly develop such lines of interest (Liu et al., 2019). Furthermore, knockout by CRISPR/Cas9 of the SlAMS gene, encoding another basic helix-loop-helix (bHLH) TF, caused downregulation leading to abnormal pollen development, which in turn decreased pollen viability and subsequently generated a male-sterile phenotype (Bao et al., 2022). Recently, other tomato CRISPR/Cas9-edited lines with male sterility phenotypes were obtained by knock-out of SlPHD_ms1 (Solyc04g008420), encoding a PHD-type TF involved in pollen formation and tapetum development, suggesting a key role for SlPHD in male sterility and aiding research into the regulatory processes of pollen and tapetum growth in tomato (Gökdemir et al., 2022). With analogous purposes, CRISPR/Cas technology was also applied in other horticultural crops, as demonstrated in cucurbit species. For example, the knockout of eIF4E by CRISPR/Cas9 in melon highlighted for the first time the association between eIF4E editing and the development of male sterility (Pechar et al., 2022). In watermelon (Citrullus lanatus L.), knockout of ClATM1 by CRISPR/Cas9 causes male sterility, confirming its self-regulatory activity and providing new insights into the molecular mechanism underlying anther development (Zhang et al., 2021b).

In monocots, several CRISPR/Cas systems for producing MS have been reported as successful applications in precision breeding. An improved CRISPR/Cas9 system was driven by the TaU3 RNA polymerase III U3 promoter, and three homologous alleles expressing the wheat redox enzyme NO POLLEN 1 (NP1) were altered to produce totally male-sterile wheat mutants (Li et al., 2020). Furthermore, with recent molecular identification of the Ms1 gene and exploiting strategies related to transgenerational gene editing (see below section 5.2), it has been possible to extend the use of the CRISPR/Cas9 system to generate Ms1 knockout wheat lines that exhibit male sterility in the first generation, demonstrating the utility of the CRISPR/Cas9 system for the rapid generation of nuclear male sterility in hexaploid species like wheat (Okada et al., 2019). Chen and colleagues created a CRISPR/Cas9 vector in maize to target the male sterility gene 8 (Ms8). The resulting mutant was male-sterile, which was compatible with Mendelian genetic rules and was stably acquired by subsequent generations (Chen et al., 2018b). Furthermore, editing ZmMTL (ZmPLA1) with the CRISPR/Cas9 system has produced maternal haploid inducers with powerful haploid identification markers useful for breeding doubled-haploid crops, such as maize itself (Dong et al., 2018). Additionally, ZmMS26, a known nuclear fertility gene (Loukides et al., 1995; Djukanovic et al., 2013) that is conserved in other monocots, like rice, wheat, and sorghum (Cigan et al., 2017), was subjected to precision editing: targeted mutagenesis of MS26 utilizing the modified I-CreI homing endonuclease or CRISPR/Cas9 resulted in the generation of new ms26 male sterile lines (Djukanovic et al., 2013; Svitashev et al., 2015; Qi et al., 2020). In rice, gene knockout by CRISPR/Cas9 of the OsHXK5 gene resulted in male sterility, contributing to demonstration that OsHXK5 contributes to a large portion of the hexokinase activity necessary for the starch utilization pathway during pollen germination and tube growth, as well as for starch biosynthesis during pollen maturation (Lee et al., 2020).

The success of CRISPR/Cas technology application has also been provided in EGMS conditions, or rather the ability to switch from fertile to sterile conditions and vice versa, by adjusting environmental variables such as temperature and photoperiod. Great progress has been recently achieved in the understanding of PGMS or TGMS traits in cereal crops, and several genes controlling P/TGMS traits have been investigated and transferred, mediating conventional breeding and/or biotechnological transformation, in specific lines on which more than 30% of cereal hybrid production depends in China (Ding et al., 2012; Zhou et al., 2012; Zhang et al., 2013; Huang et al., 2014; Zhou et al., 2014). Several studies elucidated the molecular genetic mechanisms at the base of EMGS, confirming also the interesting role assumed by phasiRNAs (phased small-interfering RNAs) generated by long-noncoding RNAs. In rice, for example, the phasiRNAs originated from PMS1T locus regulates PSMS in rice (Fan et al., 2016). Especially in rice, in the last few years, many genes influencing PGMS or TGMS traits have been discovered and cloned, and several reports describe different CRISPR/Cas-based approaches to obtain photo- and thermosensitive male-sterile lines. For example, a simple and efficient rice TGMS cultivation system using CRISPR/Cas9 editing technology was proposed to knock out the TMS5 (thermosensitive genic male-sterile 5) gene target, with great value in new commercial “transgene free” TGMS rice lines (Zhou et al., 2016). TMS5 is a nuclear recessive gene that controls the TGMS trait and extensively used in two-line hybrid rice breeding. It was the first spontaneously mutated Oryza sativa ssp. indica, identified more than 30 years ago, and encodes an RNase ZS1 endonuclease, able to degrade the temperature-sensitive ubiquitin fusion ribosomal protein L40 (UbL40) mRNA (Zhou et al., 2014). A study found that when plants were grown under a high temperature regime, several tms5 mutants developed in a background of the japonica type showed a high degree (85.3%) of pollen sterility (Zhou et al., 2016), confirming that targeted modification of TMS5 by the CRISPR/Cas9 system is a successful approach to develop TGMS lines for hybrid rice production. Huang et al. targeted the TMS5 gene, producing a mutant that was entirely male-sterile at high temperatures but male-fertile at low temperatures, with a pollen fertility transition temperature fixed at 28°C. (Huang et al., 2014). Recent studies revealed the molecular mechanism of tms5 leading to male sterility in rice to easily obtain excellent TGMS lines (Fang et al., 2022) and potentially applicable in other crops. CRISPR/Cas9-engineered mutation of TMS5 also resulted in the formation of thermosensitive male sterility in maize (Li et al., 2017). In addition, Li et al. altered the carbon starvation CSA gene in pollen grains of the rice variety ‘Kongyu 131’ and found that the csa mutant had a male-sterile phenotype in short-day and a male-fertile phenotype in long-day conditions. (i.e., photosensitive nuclear male sterile mutant) (Li et al., 2016), whereas in tomato, Shen and colleagues generated photosensitive/thermosensitive male-sterile lines by using CRISPR/Cas9 modifying the genic male-sterile 2-2 (PTGMS2-2) gene (Liu et al., 2019).

As described in the previous sections, site-specific genome editing by CRISPR/Cas9 technology is becoming a progressively more successful tool for functional, basic and applied plant research because it can generate a high rate of mutation while being relatively easy to use (Zhang et al., 2013; Lowder et al., 2015; Ma et al., 2015; Wang et al., 2016). Numerous methods have been used to create CRISPR-edited plants devoid of CRISPR constructs and other transgenes because the lack of any transgenes in gene-edited plants is a requirement for the commercialization of any CRISPR-edited plants with stable valuable traits. For public approval, gene elimination or bypassing alien elements to edit endogenous genes is fundamental and could be a strategic approach, even if transgenic intermediates are transiently necessary ( Figure 2B ). The main different strategies useful to avoid the maintenance of transgene integration have been deeply described by He and Zhao, 2020 (He and Zhao, 2020). Commonly, after CRISPR-mediated mutagenesis, the Cas9 gene and associated DNA sequences are eliminated through genetic segregation, which frequently allays public concerns about genetically modified individuals. The biggest advantage of the method is that it could allow the selection of plants that no longer contain the T-DNA sequence, producing plant materials not containing any foreign DNA even though they were produced using transgenic technology mediating stable transformation methodologies. However, the fact that many commercial crop varieties are polyploid, heterozygous, or asexually reproduced complicates these efforts. Many commercial cultivars’ genome complexity, long juvenile phase, and/or self-incompatibility limit the development of CRISPR-mediated transgenic crops since backcrossing is required to remove the CRISPR transgene.

In the past, plant transient transformation technology has been widely used as an alternative approach to facilitate rapid and efficient gene function analysis (Sheen, 2001; Chen et al., 2006). Using transient transformation methods, such as particle bombardment (Romano et al., 2003), transient transformation by Agrobacterium sp. (Cui et al., 2017) and polyethylene glycol (PEG)-mediated protoplast transfection (Cankar et al., 2022), excellent results in plant research have been achieved. Among these, the protoplast transient expression system has played a relevant role in genomics and proteomics research, resulting in a potential, rapid, and convenient technique for testing new technologies, such as GE approaches. In general, transient expression methods for protoplasts have been designed for many crop species, including monocots, dicots, herbaceous and woody species, such as rice (Yang et al., 2014a), barley (Bai et al., 2014), corn (Cao et al., 2014), apple (Maddumage et al., 2002), and grapevine (Zhao et al., 2016). These findings demonstrate the possibility and feasibility of utilizing protoplasts for CRISPR-mediated gene editing, particularly in species with a protracted juvenile phase, heterozygosity, or asexual propagation. Likewise, this strategy could represent the most feasible way to directly apply CRISPR-mediated DNA-free genome editing technologies for improving traits and increasing commercial value, as already experimentally confirmed for food and non-food crops, such as strawberry (Martin-Pizarro et al., 2019; Wilson et al., 2019), potato (Gonzalez et al., 2019; Nicolia et al., 2021; Zhao et al., 2021), lettuce (Woo et al., 2015), chicory (De Bruyn et al., 2020; Cankar et al., 2022), Nicotiana tabacum (Lin et al., 2018; Hsu et al., 2019) and Brassica oleracea (Lee et al., 2020; Hsu et al., 2021), and ornamental species, as petunia (Yu et al., 2021b). For these reasons, protoplast transient expression systems represent a promising and valid approach for generating CRISPR-edited DNA-free plant material and MS mutant production ( Figure 2B ). Numerous studies describe the different gene modification methods using transient expression of the Cas protein and associated sgRNA, mediating the main delivery methods into somatic plant cells, which may be done either as DNA vectors, through Agrobacterium infiltration (Chen et al., 2018a), or as ribonucleoprotein (RNP), using biolistic delivery (Liang et al., 2018), nanotubes (Demirer et al., 2019), virus transfection (Ellison et al., 2020), PEG-calcium (PEG–Ca²⁺) (Toda et al., 2019). Because there is no foreign DNA present during transfection, direct transfection of the RNP complex eliminates the risk of plasmid DNA insertions into the plant genome (Andersson et al., 2018). Genome editing is realizable utilizing protoplasts without the insertion of foreign CRISPR DNA and without the necessity for hybridization, introgression, or back-crossing of progeny in the T0 generation. Furthermore, protoplasts are single cells that are edited before the first cell division: new plants grow from a single modified protoplast, ensuring that all cells share the same genetic background and that edited alleles are passed down to the next generation However, RNP-mediated genome editing has been employed successfully in many plant species, targeting genes with agronomic interest, involved disease resistance (Malnoy et al., 2016), in grain yield (Toda et al., 2019), nutritional composition (Andersson et al., 2018), and male fertility (Svitashev et al., 2016). MS induction, using an analogous approach, has been successfully achieved only in maize. Svitashev and colleagues demonstrated the success of their research, in which two male fertility nuclear genes (MS26 and MS45) were targeted by purified Cas9 protein preassembled with in vitro transcribed gRNAs, demonstrating DNA-free genome editing in a major crop species using biolistically delivered Cas9–gRNA RNPs on immature embryos and subsequent plant regeneration (Svitashev et al., 2016). These positive results suggest the potential of applying similar methodologies in other large crops to increase the number of examples of male sterile lines CRISPR-edited DNA-free by transient transformation system by RNP complex.

Because CRISPR/Cas9 expression cassettes and target sites are distributed throughout the genome, segregation and deletion of CRISPR/Cas9 cassettes is conceivable through subsequent selfing or crossing ( Figure 2B ). However, in crops with a high level of genome complexity, highly heterozygous, polyploid genomes, and usually propagated vegetatively, this is not easily achieved. Specifically, efficient propagation and stacking of first-generation mutations becomes increasingly difficult or nearly impossible with polyploidy.

Numerous new strategies have been developed to extend the CRISPR toolbox, and many of these new schemes could also take advantage from transgenerational gene editing (TGE)-based strategies, defined as the continued ability of Cas9 to edit also after cross: this means that if the Cas9 nuclease is still active, after cross it will encounter a new WT allele, which can be edited to create independent alleles. TGE has been utilized for a variety of applications, some of which are not always defined as TGE, such as the editing new alleles in polyploid crops, the creating allelic variation, and the editing target genes in refractory genetic backgrounds (Impens et al., 2022). Mutations are frequently found only in a fraction of the homoeoalleles targeted by the same sgRNA in polyploid crops such as hexaploid common wheat (Triticum aestivum) and tetraploid cotton (Gossypium hirsutum) (Wang et al., 2018a; Wang et al., 2018b; Wang et al., 2018c). While expressing CRISPR/Cas9 for more than one generation during TGE promotes on-target homoeoallele editing, it does not always boost off-targeting.

On the basis of TGE, with the purpose of accelerating the understanding of MS and ensuring speedy improvement, a new approach (Ramadan et al., 2021) was tested for example in cotton system, in which the use of pooled sgRNAs targeting single or duplicated genes belonging to different families provided a large number of intentional mutants that would help us know male sterility in cotton itself. Furthermore, this strategy ensured a rapid characterization of the key genes which may influence fertility in cotton, with important consequences for cotton future genetic improvement (Ramadan et al., 2021). Furthermore, as previously mentioned, a TGE-based methodology was implemented to facilitate the ongoing modification of homoeoalleles in species like hexaploid wheat, which is not easily amenable to conventional mutagenesis techniques. In this approach, a transgenic line may exhibit modifications in only a subset of homoeoalleles. However, through self-crossing and careful selection of plants inheriting the T-DNA, it becomes possible to modify all homoeoalleles. Singh and colleagues proposed an effective utilization of the CRISPR/Cas system and next-generation sequencing for mutant analysis in wheat. They successfully established the role of TaMs26 in wheat pollen generation by combining mutations in TaMs26 from the A-, B-, and D-genomes through crossing, resulting in the development of male sterile plants (Singh et al., 2017). Orthologous Ms26 mutations in rice and sorghum plants, as in maize, confer a recessive male sterile phenotype, and restoration of fertility in these mutant sorghum plants was achieved by a copy of maize Ms26 (Cigan et al., 2017). Afterwards, with recent molecular identification of the male fertility Ms1 gene, it has been possible to extend the use of the CRISPR/Cas9 system to generate Ms1 knockout wheat lines with male sterility in the first generation, demonstrating the potential of the CRISPR/Cas9 system for the fast generation of GMS in hexaploid wheat (Singh et al., 2018; Okada et al., 2019).

This evidence on transgenerational gene editing activity demonstrates that TGE can contribute to novel variation in the offspring of CRISPR/Cas9-expressing plants, and that Cas9-inducible trait can be transferred by crossing the plants expressing the gene editing constructs with the lines of interest.