5.1 Development strategies of the BMS systems

The development strategies of BMS systems based on GMS genes and other new technologies have been reviewed thoroughly and systemically in our laboratory [3]. In general, there are two kinds of strategies to develop BMS systems that have an application potential in hybrid seed production: transgenic construct-driven nontransgenic seed systems and transgenic male-sterility systems. The former includes the SPT and MCS systems in maize, SPT-like systems in maize, rice, and wheat,


#### Table 2. Strategy and application assessment of BMS systems in crop plants.

Molecular Cloning of Genic Male-Sterility Genes and Their Applications for Plant Heterosis… DOI: http://dx.doi.org/10.5772/intechopen.86976

while the latter includes the RHS1 and RHS2 systems in maize and Barnase/Barstar system in oilseed rape (Table 2).

## 5.1.1 SPT- and SPT-like systems in crops

anther and pollen development in crop plants and have provided an important basis for developing BMS lines. Several attempts have been made to utilize GMS genes in combination with new technologies to achieve more feasible BMS systems in crop plants [3, 4, 6, 100]. Here, we introduce briefly the strategies, assessment, and

The development strategies of BMS systems based on GMS genes and other new technologies have been reviewed thoroughly and systemically in our laboratory [3]. In general, there are two kinds of strategies to develop BMS systems that have an application potential in hybrid seed production: transgenic construct-driven nontransgenic seed systems and transgenic male-sterility systems. The former includes the SPT and MCS systems in maize, SPT-like systems in maize, rice, and wheat,

MCS Maize 1. Genetic stability and

SPT Maize 1. Genetic stability of SPT

Crop Application assessment Application

heterosis of ms line 2. Genetic stability analysis of MCS maintainer lines

maintainer lines 2. SPT transgene transmission rate through pollen

Maize 1. Ms44 plants increase kernel number 2. Ms44 hybrids showed no yield penalty

Rice The osnp1 line was crossed with 1200 rice germplasms, 10% out-yielded the best local cultivars

Maize 1. The consistency in

RHS2 Maize 1. The endogenous mts-

performance across inbreds 2. The RHS inbreds showed no yield penalty

siRNAs are present widely 2. The elite event was introgressed into 15 maize germplasms

1. It is feasible in some crop plants 2. Double female parent seeds needed

Wheat No data No data [49]

status

Commercial application in maize

Product test [9, 18, 21]

Product test [22]

Product test [69]

Commercial application in maize

Commercial application in canola

Product test [102]

Ref.

[62]

[101]

[103, 104]

applications of some typical BMS systems in crop plants (Table 2).

5.1 Development strategies of the BMS systems

Synthetic Biology - New Interdisciplinary Science

system

SPT-like (based on dominant ms44 gene)

SPT-like (based on OsNP1 gene)

SPT-like (based on TaMs1 gene)

RHS1: glyphosatemediated male sterility

> Barnase/ Barstar system

Strategy and application assessment of BMS systems in crop plants.

Oilseed rape

Strategy BMS

Transgenic construct-driven non-transgenic product systems

Transgenic malesterility systems

Table 2.

88

The SPT system is one of the representatives of transgenic construct-driven nontransgenic seed systems initially developed in maize by using a transgenic maintainer strategy [62]. The SPT transgenic maintainer line is created by transforming the plant of interest with an SPT construct consisting of three components: (i) a wild-type male-fertility gene (e.g., Ms45) to restore fertility, (ii) a pollen lethality gene (e.g., ZmAA) to disrupt normal pollen development, and (iii) a fluorescent seed color marker gene (e.g., DsRed2) for seed sorting. Of the pollen grains produced by the SPT maintainer line, all have the ms45 genotype, 50% are nontransgenic, and 50% have the SPT transgenic elements. The latter grains are unable to germinate due to expression of the ZmAA gene. Thus, self-pollination of the SPT maintainer line produces both seeds with the same genotype of the SPT maintainer line (ms45/ms45 + SPT-T-DNA) and seeds with the male-sterile genotype (ms45/ ms45). The two types of seeds can be efficiently separated by mechanical color sorting, since the 50% of seeds contain the SPT elements showing a red color under green excitation light. When the male-sterile line (ms45/ms45) is pollinated with the SPT maintainer line, almost 100% of the resulting seeds have the ms45/ms45 genotype and can be used as male-sterile female lines for crossbreeding and hybrid seed production.

Since the maize SPT system was developed and applied successfully [62], several SPT-like systems have been developed in maize, rice, and wheat based on ZmMs44, OsNP1, and TaMs1 genes, respectively [22, 49, 69]. Although there are potentially many advantages of the SPT system, the rate of transgene transmission through pollen was found to vary with the highest rate being 0.518% [62]. Therefore, there is an increased risk of transgenic pollen flow during the male-sterile line propagation phase, thus resulting in greatly limited application in the countries and regions with strict biotechnology regulatory oversight.

#### 5.1.2 MCS System

To decrease the rate of transgene transmission through the pollen of SPT maintainer lines, our laboratory developed a MCS system by transforming a single MCS construct into the maize ms7, ms30, or ms33 mutant [9, 105, 106]. The MCS construct contains five functional modules: (i) a male-fertility gene (e.g., ZmMs7, ZmMs30 or ZmMs33) to restore fertility, (ii) two pollen disruption genes (e.g., ZmAA and Dam) to disrupt the production of transgenic pollen, (iii) a fluorescent color marker gene (e.g., DsRed2 or mCherry) for seed color sorting, and (iv) an herbicide-resistant gene (e.g., Bar) to prevent sophistication of seeds because it is beneficial for the propagation of high-purity MCS transgenic maintainer line seeds through herbicide spraying during specific stages of production. As the MCS construct harbors two pollen disruption modules, both of which can inhibit transgenic pollen formation or function, the transgene transmission rate through pollen is greatly decreased. Furthermore, the Bar gene in the MCS construct is helpful for propagating highly pure seeds of the transgenic maintainer line. Compared with the SPT construct, the MCS construct, which harbors two additional functional modules, the Bar and Dam genes, can produce maintainer and male-sterile lines with higher purity and greatly decrease the transgene transmission rate as well as the risk of transgene flow in commercial maize hybrid seed production. To promote commercial application, a field test of the MCS system in China is currently underway.

As described above, although the final product of transgenic construct-driven male-sterility systems is non-transgenic, the use of these systems is often limited by the lack of GMS mutants and male-fertile genes in many crops. Consequently, many biotechnology strategies have been developed in the past 30 years to produce artificially dominant male-sterile plants independent of GMS mutants and malefertility genes, such as the RHS systems in maize and Barnase/Barstar system in oilseed rape.

the pollen of transgenic SPT maintainer to pollinate non-transgenic plants. Then ears were harvested from the non-transgenic female parent plants and examined under visible light for the presence of pink seeds expressing the DsRed2 protein. The transgene transmission rate through pollen varied from 0% to 0.518% with different constructs and transformants. As the transformant DP-32138-1 showed the lowest transgene transmission rate that was maintained across generations and in different inbred backgrounds, it was selected as the SPT maintainer line for use in

Molecular Cloning of Genic Male-Sterility Genes and Their Applications for Plant Heterosis…

The RHS system was developed by minimizing the CP4-EPSPS expression in the tassel and maximizing glyphosate delivery to the tassel resulting in consistent male sterility for hybrid seed corn production [101]. Therefore, the glyphosate spray timing, dose, target, and mode should be examined firstly in different maize varieties. Subsequently, the consistency in performance of the RHS technology across a broad range of inbred varieties is essential and has been examined in field trials. The performance standard for RHS is 0.5% anther extrusion or >99.5% tassel sterility to insure high purity in hybrid seeds. The field data show that RHS has consistently surpassed the performance standard since 2007 when examined across an increasing number of inbred varieties. In fact, in 2011 and 2012, 100% sterility or zero anther extrusion was observed across 46 and 47 inbred varieties, respectively. The RHS inbreds have shown comparable yield to mechanical detasseling; furthermore, the F1 hybrid seeds produced by crossing RHS with RR showed full resistance to glyphosate and comparable performance to hybrid seeds produced by mechanical

The Barnase/Barstar system was the first BMS system developed in tobacco and oilseed rape plants based on tapetal cell-specific expression of TA29-barnase and TA29-barstar genes [103, 104]. TA29-barnase transgene leads to dominant male sterility due to selective destruction of anther tapetal cells, while TA29-barstar transgene can suppress the expression of TA29-barnase transgene and restore the male fertility. The feasibility of this system was confirmed in many crop plants, such as oilseed rape, tobacco, lettuce, chicory, cauliflower, tomato, cotton, and maize. However, the dominant male-sterility line must maintained in heterozygous plants by crossing with a wild-type line in the same (isogenic) genetic background. The elimination of the fertile segregants by herbicide spray doubles the amount of female parent seed needed for hybrid seed production, which could limit the applicability of the system in crops with low multiplication factor and relatively low

As shown in Table 2, there are only three BMS systems that have been applied in commercial hybrid seed production, including SPT, RHS, and Barnase/Barstar systems. The SPT system has been deregulated by USDA APHIS in 2011 and is thus available for commercial hybrid seed production in maize [110]. Unlike other BMS systems, the maize inbred parent lines produced using the SPT system do not inherit SPT transgene from the SPT maintainer line and thus are non-transgenic. Furthermore, both the commercial maize hybrid seed produced from the SPT system and the resulting commodity maize grain harvested from these hybrid plants are non-transgenic. Subsequently, acknowledgement of the non-transgenic status of progeny produced by the SPT system is also supported by regulatory agencies in Australia and Japan [111, 112]. Therefore, hybrid maize and commodity grain produced from the SPT system are non-genetically modified (non-GM) and subject only to those regulations applicable to conventional non-GM maize. The performance of RHS system has been evaluated by Monsanto manufacturing group

maize male-sterile parent seed increase [62].

DOI: http://dx.doi.org/10.5772/intechopen.86976

detasseling [101].

plant density [4].

91

5.3 Commercial application of the BMS systems

#### 5.1.3 RHS system

The RHS system, which is based on glyphosate-mediated male sterility, is deployed for hybrid seed production by Monsanto [101]. The RHS system consists of RHS and RR transgene constructs. The RHS transgene cassette contains the CP4- EPSPS gene (encoding 5-enolpyruvyl-shikimate 3-phosphate synthase, which is insensitive or resistant to glyphosate) driven by the enhanced 35S promoter, which has been shown to be poorly expressed in tapetum cells and microspores, and thus the resulting RHS plant demonstrates male sterility following glyphosate application with little/no injury to the rest of the plant. The RR transgene construct comprises a double expression cassette providing high constitutive expression of CP4-EPSPS resulting in robust resistance to glyphosate. In hybrid seed production fields, rows of an RHS female line are interplanted with rows of an RR male line, and over-the-top sprays of glyphosate induce male sterility in RHS female plants, which are subsequently pollinated by RR male plants. By withholding glyphosate, the RHS plants remain fully fertile and are capable of self-pollination for propagation of female line without the need for a maintainer line.

The RHS system replaces mechanical detasseling with glyphosate spray and greatly simplifies the process of hybrid seed corn production. Recently, this system has been improved as RHS2 by using endogenous maize male tissue-specific small interfering RNAs to trigger cleavage of the CP4-EPSPS mRNA specifically in tassels, resulting in glyphosate-sensitive male cells [102].

#### 5.1.4 Barnase/Barstar system

The Barnase/Barstar system was the first dominant BMS system developed in rapeseed and tobacco [103, 104] and then has been tested and tried in wheat and rice [107, 108]. The barnase and barstar genes are fused with the tapetum-specific TA29 promoter and then transformed individually into plants. The TA29-barnase transformed plants are completely male-sterile and are crossed with TA29-barstarexpressing fertile plants, which results in the co-expression of barnase and barstar genes in the anther tapetal cell layer. The inactivation of barnase by barstar leads to the restoration of fertility in the hybrid F1 plants [109].

#### 5.2 Application assessment of the BMS systems

Although serval BMS systems had been developed during the past decades, only three of them were thoroughly assessed and applied in commercial hybrid seed production in some crops, such as SPT, RHS, and Barnase/Barstar systems.

In SPT system, the stability of transgenes in prospective SPT maintainer lines were examined based on Southern blot analyses of genomic DNA from T0 to T4 plants to assess the integration and structural fidelity of the transgenes. No changes in hybridization patterns with three different restriction enzyme digestions of the SPT transgenes indicated that the transgenes are stable over multiple generations. More importantly, the transgene transmission through pollen was tested by using

#### Molecular Cloning of Genic Male-Sterility Genes and Their Applications for Plant Heterosis… DOI: http://dx.doi.org/10.5772/intechopen.86976

the pollen of transgenic SPT maintainer to pollinate non-transgenic plants. Then ears were harvested from the non-transgenic female parent plants and examined under visible light for the presence of pink seeds expressing the DsRed2 protein. The transgene transmission rate through pollen varied from 0% to 0.518% with different constructs and transformants. As the transformant DP-32138-1 showed the lowest transgene transmission rate that was maintained across generations and in different inbred backgrounds, it was selected as the SPT maintainer line for use in maize male-sterile parent seed increase [62].

The RHS system was developed by minimizing the CP4-EPSPS expression in the tassel and maximizing glyphosate delivery to the tassel resulting in consistent male sterility for hybrid seed corn production [101]. Therefore, the glyphosate spray timing, dose, target, and mode should be examined firstly in different maize varieties. Subsequently, the consistency in performance of the RHS technology across a broad range of inbred varieties is essential and has been examined in field trials. The performance standard for RHS is 0.5% anther extrusion or >99.5% tassel sterility to insure high purity in hybrid seeds. The field data show that RHS has consistently surpassed the performance standard since 2007 when examined across an increasing number of inbred varieties. In fact, in 2011 and 2012, 100% sterility or zero anther extrusion was observed across 46 and 47 inbred varieties, respectively. The RHS inbreds have shown comparable yield to mechanical detasseling; furthermore, the F1 hybrid seeds produced by crossing RHS with RR showed full resistance to glyphosate and comparable performance to hybrid seeds produced by mechanical detasseling [101].

The Barnase/Barstar system was the first BMS system developed in tobacco and oilseed rape plants based on tapetal cell-specific expression of TA29-barnase and TA29-barstar genes [103, 104]. TA29-barnase transgene leads to dominant male sterility due to selective destruction of anther tapetal cells, while TA29-barstar transgene can suppress the expression of TA29-barnase transgene and restore the male fertility. The feasibility of this system was confirmed in many crop plants, such as oilseed rape, tobacco, lettuce, chicory, cauliflower, tomato, cotton, and maize. However, the dominant male-sterility line must maintained in heterozygous plants by crossing with a wild-type line in the same (isogenic) genetic background. The elimination of the fertile segregants by herbicide spray doubles the amount of female parent seed needed for hybrid seed production, which could limit the applicability of the system in crops with low multiplication factor and relatively low plant density [4].

#### 5.3 Commercial application of the BMS systems

As shown in Table 2, there are only three BMS systems that have been applied in commercial hybrid seed production, including SPT, RHS, and Barnase/Barstar systems. The SPT system has been deregulated by USDA APHIS in 2011 and is thus available for commercial hybrid seed production in maize [110]. Unlike other BMS systems, the maize inbred parent lines produced using the SPT system do not inherit SPT transgene from the SPT maintainer line and thus are non-transgenic. Furthermore, both the commercial maize hybrid seed produced from the SPT system and the resulting commodity maize grain harvested from these hybrid plants are non-transgenic. Subsequently, acknowledgement of the non-transgenic status of progeny produced by the SPT system is also supported by regulatory agencies in Australia and Japan [111, 112]. Therefore, hybrid maize and commodity grain produced from the SPT system are non-genetically modified (non-GM) and subject only to those regulations applicable to conventional non-GM maize. The performance of RHS system has been evaluated by Monsanto manufacturing group

As described above, although the final product of transgenic construct-driven male-sterility systems is non-transgenic, the use of these systems is often limited by the lack of GMS mutants and male-fertile genes in many crops. Consequently, many biotechnology strategies have been developed in the past 30 years to produce artificially dominant male-sterile plants independent of GMS mutants and malefertility genes, such as the RHS systems in maize and Barnase/Barstar system in

The RHS system, which is based on glyphosate-mediated male sterility, is deployed for hybrid seed production by Monsanto [101]. The RHS system consists of RHS and RR transgene constructs. The RHS transgene cassette contains the CP4- EPSPS gene (encoding 5-enolpyruvyl-shikimate 3-phosphate synthase, which is insensitive or resistant to glyphosate) driven by the enhanced 35S promoter, which has been shown to be poorly expressed in tapetum cells and microspores, and thus the resulting RHS plant demonstrates male sterility following glyphosate application with little/no injury to the rest of the plant. The RR transgene construct comprises a double expression cassette providing high constitutive expression of CP4-EPSPS resulting in robust resistance to glyphosate. In hybrid seed production fields, rows of an RHS female line are interplanted with rows of an RR male line, and over-the-top sprays of glyphosate induce male sterility in RHS female plants, which are subsequently pollinated by RR male plants. By withholding glyphosate, the RHS plants remain fully fertile and are capable of self-pollination for propaga-

The RHS system replaces mechanical detasseling with glyphosate spray and greatly simplifies the process of hybrid seed corn production. Recently, this system has been improved as RHS2 by using endogenous maize male tissue-specific small interfering RNAs to trigger cleavage of the CP4-EPSPS mRNA specifically in tassels,

The Barnase/Barstar system was the first dominant BMS system developed in rapeseed and tobacco [103, 104] and then has been tested and tried in wheat and rice [107, 108]. The barnase and barstar genes are fused with the tapetum-specific TA29 promoter and then transformed individually into plants. The TA29-barnase transformed plants are completely male-sterile and are crossed with TA29-barstarexpressing fertile plants, which results in the co-expression of barnase and barstar genes in the anther tapetal cell layer. The inactivation of barnase by barstar leads to

Although serval BMS systems had been developed during the past decades, only

In SPT system, the stability of transgenes in prospective SPT maintainer lines were examined based on Southern blot analyses of genomic DNA from T0 to T4 plants to assess the integration and structural fidelity of the transgenes. No changes in hybridization patterns with three different restriction enzyme digestions of the SPT transgenes indicated that the transgenes are stable over multiple generations. More importantly, the transgene transmission through pollen was tested by using

three of them were thoroughly assessed and applied in commercial hybrid seed production in some crops, such as SPT, RHS, and Barnase/Barstar systems.

tion of female line without the need for a maintainer line.

resulting in glyphosate-sensitive male cells [102].

the restoration of fertility in the hybrid F1 plants [109].

5.2 Application assessment of the BMS systems

5.1.4 Barnase/Barstar system

90

oilseed rape.

5.1.3 RHS system

Synthetic Biology - New Interdisciplinary Science

which produces the hybrid seeds that are sold in the marketplace. A few modifications have been implemented to make the RHS system more practical and manageable in the field [101]. Most recently, the second-generation RHS (RHS2) technology combines the relative simplicity and convenience of a systemic herbicide spray methodology with targeted protein expression to create an inducible male sterility system for industrial production of maize hybrid seeds in an environmentally independent manner [102]. The Barnase/Barstar system has been used successfully for the commercial production of canola hybrids (Brassica napus) in Canada [4]. However, most of the BMS systems have not been used in commercial hybrid seed production, maybe because of lacking the cost-effective and environment-friendly BMS systems and/or the regulatory acceptance of using BMS systems among different countries.

transgenic maintainer line based on completely male-sterile mutants and the male-fertility genes and requirement of fluorescent seed color-sorting machine. Therefore, the transgenic male-sterility systems, such as RHS system based on glyphosate-mediated male sterility, have also been developed and used in commercial hybrid seed production in maize [101, 102]. This system is independent of male-sterility mutants and male-fertility genes, no need for transgenic maintainer line and seed color sorting, and the herbicide-resistant male-sterility lines are helpful to highly efficient and mechanized hybrid seed production. However, this transgenic male-sterility system is limited by the "zero tolerance" regulatory policy preventing transgenic planting in many countries, need for application of herbicide in hybrid seed production fields, and potential risk of gene flow. In summary, both SPT and RHS systems have advantages and disadvantages compared with

DOI: http://dx.doi.org/10.5772/intechopen.86976

Molecular Cloning of Genic Male-Sterility Genes and Their Applications for Plant Heterosis…

With the advance of molecular cloning methods including both forward and reverse genetic approaches, especially the next-generation sequencing technology and genome-editing technology (e.g., CRISPR-Cas9), more GMS genes in cereal crops with large and complex genome (e.g., wheat and barley) will be identified and characterized by using multiple strategies as described in this chapter. At the same time, the application value of the putative GMS genes should be assessed systemically in genetic stability of male sterility, effects on heterosis performance, and potential linkage with detrimental traits. This will not only boost our understanding in the molecular mechanism of anther and pollen development but also give great opportunity to develop novel BMS systems for commercial hybrid seed production

The research in our lab was supported by the National Transgenic Major Program of China (2018ZX0800922B, 2018ZX0801006B), the National Natural Science Foundation of China (31771875, 31871702), the National Key Research and

2017YFD0101201), the Fundamental Research Funds for the Central Universities of China (06500060), and the "Ten Thousand Plan"—National High-Level Talents

Development Program of China (2018YFD0100806, 2017YFD0102001,

The authors declared that they have no conflict of interest.

CRISPR clustered regularly interspersed short palindromic repeats

each other.

in cereal crops.

Acknowledgements

Conflict of interest

Abbreviations

93

Special Support Plan (For Xiangyuan Wan).

BMS biotechnology-based male sterility

EGMS environment-sensitive genic male sterility

BSA bulked segregant analysis CMS cytoplasmic male sterility

EMS methanesulfonate EST expressed sequence tag GM genetically modified GMS genic male sterility
