**1. Introduction**

The use of hybrid greatly increased rice production worldwide due to the improved yields, better tolerance to pest, diseases and environmental stress compared to inbred varieties. The discovery of cytoplasmic male sterility was the major milestone to the development of hybrid rice [1]. Further discovery of two line male sterility made hybrid breeding more efficient and further increased the probability in finding the best performing hybrid combinations [2].

Two different male sterility systems are available for hybrid seed production (**Figure 1**). The first is a cytoplasmic male sterility (CMS) which is a three-line system that uses a male sterile line, a restorer line and a maintainer line. The male sterility is more stable albeit more complicated to breed and maintain [3]. The second is the two-line male sterility system that uses a genetic male sterile which is controlled by temperature, photoperiod or both. The use of this system is increasing due to the ease in breeding, finding more heterotic combinations, and in seed multiplication of parental male sterile lines. However, hybrid seed production may

**Figure 1.**

*Comparison of two-line and three-line hybrid rice breeding system. A: two line hybrid system; S—genetic male sterile, R—restorer/pollen fertile, F1—hybrid. B: three line hybrid system A—CMS line, B—maintainer line, R—restorer line with restorer gene, F1—hybrid.*

be catastrophic if there are severe changes in environmental conditions [4]. Both systems proved effective in hybrid rice production which increased yields by up to 20% therefore, increased farm profitability and has contributed significantly in addressing global food security.

Heterosis in hybrid rice minimize the impact of reduced yields brought by diseases compared to the inbred counterpart. However, due to narrow genetic diversity of the male sterile parent, they became vulnerable to pathogens and pests resulting to the loss of its yield potential [5]. This makes it difficult for growers to recover the high cost of seed and F1 production. It became apparent that discovering new sources of male sterility to increase genetic diversity and further introgression of resistance genes are necessary to secure the yield gain in hybrid rice [6].

This chapter focuses on the discovery of rice male sterility, genetics, mechanisms and procedures in multiplication and handling of male sterile rice for hybrid rice breeding.

### **2. Cytoplasmic male sterile (CMS) rice lines**

Development and cultivation of hybrid rice started in China with the initial work of rice breeder Yuan Longping. As early as 1964, Yuan Longping have tested different male sterile lines however, no stable sterility exists and the group started resorting to making distant hybridization by crossing wild rice with cultivated rice. In 1970, a wild-abortive type cytoplasmic male sterile rice CMS-WA were discovered which eventually leads to the release of the first hybrid rice in 1976. By 1980's, hybrid rice accounts to about 55% of the total rice planting area in China [7, 8]. More CMS types were discovered that further expand the diversity in hybrid rice three-line system. These were developed by direct crossing or backcross breeding from two different species, subspecies or different cultivars [9]. The major type of CMS systems with their cytoplasm and nucleus sources are shown in **Table 1**.

**15**

*Genetics and Breeding System for Cytoplasmic and Genetic Male Sterility in Rice*

There were more than 60 types of CMS systems discovered in China alone but most of them may only be classified in three types CMS-BT (Boro II), CMS-WA (wild abortive), and CMS-HL (Honglian) [10, 11]. The three major types produces pollen that lack starch or are starch deficient while CMS-LD and CMS-CW produces morphologically normal pollen grains but were unable to fully germinate [12]. In CMS-WA, pollen abortion occur at a uninucleate stage primarily during microspore development [13]. The result is an irregularly shaped and lightly stained pollen when treated with 1% iodine potassium iodide solution (I2KI). The genotype of sporophytic tissues determines pollen abortion. In CMS-BT, pollen abortion occurs at trinucleate stage with pollen lightly stained due to deficiency of starch and spherical in shape rather than irregular [14]. In CMS-HL, pollen abortion appears at binucleate stage and the pollen is spherical in shape but without starch. Restoration to fertility in all CMS type except CMS-WA are all gametophytic therefore produc-

**Cytoplasm Nucleus**

CMS-BT Chinsurah Boro II (*indica*) Liming (*japonica*) CMS-HL Hong lian (*Oryza rufipogon*) Liantanzao (*indica*) CMS-CW Chinese wild W1 (*Oryza rufipogon*) Reimei (*japonica*) CMS-WA Wild abortive (*Oryza rufipogon*) Erjiunan (*indica*) CMS-LD Burmese "Lead rice" (*indica*) Fujisaka 5 (*japonica*)

ing half of the pollen fertile in the F1 generation (**Figure 2**).

crossed to B line restores fertility in the F1 (**Figure 3**) [15].

specific cytoplasmic factor is still unknown [17].

**2.1 Genetics and mechanism of cytoplasmic male sterility (CMS)**

Sterility in CMS is controlled by the interaction of genes in the cytoplasm and the nucleus. The sterility factor S is located in the mitochondrial DNA while the *rf* (restorer of fertility) allele is located in the nucleus. The plant is sterile (A line) if it carries both the S factor and the recessive allele *rf*. Maintainer line (B line) carries the *rf* allele but has a different cytoplasmic factor N which allows the plant to be fertile. The B-line has the ability to make the S line produce seeds after crossing but the progeny remains sterile thus useful for S line seed multiplication. Restorers (R line) is the diverse pollen fertile parent that carries the dominant *Rf* gene that when

CMS-BT genes were the first to be identified that has the mitochondrial open

CMS-HL carries the mitochondrial *atp6-orfH79* in which *orfH79* and *orf79* are 98% identical in DNA sequence [9]. In CMS-WA *orf224*, *orf284* and *orf288* were discovered with one still unknown segment. Together they encode a 325-residue protein with three transmembrane segments that are believed to be responsible for CMS trait [17]. A *B-atp-orf79* like structures were also found in CMS-LD that may be link to male sterility but in CMS-CW, no similar structures were identified, thus the

A total of six restorer of fertility genes (*Rf*) in rice have been discovered. These are *Rf1a*, *Rf1b*, *Rf2*, *Rf4*, *Rf5*, and *Rf17*. These genes were classified as pentatricopeptide repeat (PPR) proteins which are RNA binding and act in post-transcriptional mRNA process in cell organelles [18]. *Rf1a* and *Rf1b* restores fertility in CMS-BT while in CMS-HL, 50% fertile pollen can be restored by either *Rf5* or *Rf6*. If both present in

reading frame *orf79* [16] and is co-transcribed with *B-atp-orf79*. Similarly,

*DOI: http://dx.doi.org/10.5772/intechopen.85191*

**MS type Male sterility source**

*Primary CMS male sterility systems utilized in hybrid rice production.*

**Table 1.**

*Genetics and Breeding System for Cytoplasmic and Genetic Male Sterility in Rice DOI: http://dx.doi.org/10.5772/intechopen.85191*


### **Table 1.**

*Protecting Rice Grains in the Post-Genomic Era*

addressing global food security.

*R—restorer line with restorer gene, F1—hybrid.*

rice breeding.

**Figure 1.**

be catastrophic if there are severe changes in environmental conditions [4]. Both systems proved effective in hybrid rice production which increased yields by up to 20% therefore, increased farm profitability and has contributed significantly in

*Comparison of two-line and three-line hybrid rice breeding system. A: two line hybrid system; S—genetic male sterile, R—restorer/pollen fertile, F1—hybrid. B: three line hybrid system A—CMS line, B—maintainer line,* 

Heterosis in hybrid rice minimize the impact of reduced yields brought by diseases compared to the inbred counterpart. However, due to narrow genetic diversity of the male sterile parent, they became vulnerable to pathogens and pests resulting to the loss of its yield potential [5]. This makes it difficult for growers to recover the high cost of seed and F1 production. It became apparent that discovering new sources of male sterility to increase genetic diversity and further introgression

This chapter focuses on the discovery of rice male sterility, genetics, mechanisms and procedures in multiplication and handling of male sterile rice for hybrid

Development and cultivation of hybrid rice started in China with the initial work of rice breeder Yuan Longping. As early as 1964, Yuan Longping have tested different male sterile lines however, no stable sterility exists and the group started resorting to making distant hybridization by crossing wild rice with cultivated rice. In 1970, a wild-abortive type cytoplasmic male sterile rice CMS-WA were discovered which eventually leads to the release of the first hybrid rice in 1976. By 1980's, hybrid rice accounts to about 55% of the total rice planting area in China [7, 8]. More CMS types were discovered that further expand the diversity in hybrid rice three-line system. These were developed by direct crossing or backcross breeding from two different species, subspecies or different cultivars [9]. The major type of CMS systems with their cytoplasm and nucleus sources are shown in **Table 1**.

of resistance genes are necessary to secure the yield gain in hybrid rice [6].

**2. Cytoplasmic male sterile (CMS) rice lines**

**14**

*Primary CMS male sterility systems utilized in hybrid rice production.*

There were more than 60 types of CMS systems discovered in China alone but most of them may only be classified in three types CMS-BT (Boro II), CMS-WA (wild abortive), and CMS-HL (Honglian) [10, 11]. The three major types produces pollen that lack starch or are starch deficient while CMS-LD and CMS-CW produces morphologically normal pollen grains but were unable to fully germinate [12]. In CMS-WA, pollen abortion occur at a uninucleate stage primarily during microspore development [13]. The result is an irregularly shaped and lightly stained pollen when treated with 1% iodine potassium iodide solution (I2KI). The genotype of sporophytic tissues determines pollen abortion. In CMS-BT, pollen abortion occurs at trinucleate stage with pollen lightly stained due to deficiency of starch and spherical in shape rather than irregular [14]. In CMS-HL, pollen abortion appears at binucleate stage and the pollen is spherical in shape but without starch. Restoration to fertility in all CMS type except CMS-WA are all gametophytic therefore producing half of the pollen fertile in the F1 generation (**Figure 2**).

### **2.1 Genetics and mechanism of cytoplasmic male sterility (CMS)**

Sterility in CMS is controlled by the interaction of genes in the cytoplasm and the nucleus. The sterility factor S is located in the mitochondrial DNA while the *rf* (restorer of fertility) allele is located in the nucleus. The plant is sterile (A line) if it carries both the S factor and the recessive allele *rf*. Maintainer line (B line) carries the *rf* allele but has a different cytoplasmic factor N which allows the plant to be fertile. The B-line has the ability to make the S line produce seeds after crossing but the progeny remains sterile thus useful for S line seed multiplication. Restorers (R line) is the diverse pollen fertile parent that carries the dominant *Rf* gene that when crossed to B line restores fertility in the F1 (**Figure 3**) [15].

CMS-BT genes were the first to be identified that has the mitochondrial open reading frame *orf79* [16] and is co-transcribed with *B-atp-orf79*. Similarly, CMS-HL carries the mitochondrial *atp6-orfH79* in which *orfH79* and *orf79* are 98% identical in DNA sequence [9]. In CMS-WA *orf224*, *orf284* and *orf288* were discovered with one still unknown segment. Together they encode a 325-residue protein with three transmembrane segments that are believed to be responsible for CMS trait [17]. A *B-atp-orf79* like structures were also found in CMS-LD that may be link to male sterility but in CMS-CW, no similar structures were identified, thus the specific cytoplasmic factor is still unknown [17].

A total of six restorer of fertility genes (*Rf*) in rice have been discovered. These are *Rf1a*, *Rf1b*, *Rf2*, *Rf4*, *Rf5*, and *Rf17*. These genes were classified as pentatricopeptide repeat (PPR) proteins which are RNA binding and act in post-transcriptional mRNA process in cell organelles [18]. *Rf1a* and *Rf1b* restores fertility in CMS-BT while in CMS-HL, 50% fertile pollen can be restored by either *Rf5* or *Rf6*. If both present in

#### **Figure 2.**

*A schematic presentation of the five well-studied rice CMS types. Abbreviations for cytoplasm sources are RWA for wild-abortive Oryza rufipogon, RRA for red-awned O. rufipogon, and RW1 for Chinese wild rice (O. rufipogon) accession W1; IBT and ILD for indica Boro-II type and Lead rice, respectively. Nucleus sources are either indica (I) or japonica (J) [7].*

### **Figure 3.**

*Schematic diagram of CMS three line system. Rfrf = nuclear gene homozygote recessive, RfRf = nuclear gene homozygote dominant, Rfrf = nuclear gene heterozygous, N = cytoplasmic factor (fertile), s = cytoplasmic factor (sterile).*

CMS-HL, *Rf5* and *Rf6* restores 75% of fertile pollen in the F1. Further analysis by sequencing and cloning concluded that *Rf1* is the same gene as *Rf5* located in chromosome 10 [19]. *Rf3* and *Rf4* restores fertility in CMS-WA and are located in chromosome 1 and 10 respectively with the latter recently cloned [20]. CMS-LD fertility can be restored by *Rf1* or *Rf2* while CMS-CW can only be restored by a single gene *Rf17* [12].

### **2.2 Breeding and diversification of CMS sources**

Diversification of both CMS maintainers and restorer lines are very important to guarantee the continued progress of finding the best hybrid combinations.

**17**

**3.1 Genetics of male sterility**

*Genetics and Breeding System for Cytoplasmic and Genetic Male Sterility in Rice*

Extensive evaluation of lines and backcross breeding were employed to improve the lines and adapt to a particular environment [21]. Furthermore, new maintainers and restorers were developed from the original donors. A new CMS source was discovered in Dongxiang wild rice by continuous backcrossing to the *indica* variety Zongzao 35 [10]. In another study, a new source was found from interspecific cross of an *indica* with an African rice (*Oryza glaberrima Steud.*) that showed similarity to

Although the CMS three-line system greatly increased yields in hybrid rice, there are difficulties and limitations on its use. One of the difficulties is the need to simultaneously develop maintainer lines (B lines) by subsequent nucleus substitution of the original CMS line with the B lines through repeated backcrossing. Furthermore, there are also limited choices available for restorer lines (R lines) with only about 5% of the current existing lines can be used that carries the restorer gene [7]. The discovery of genetic male sterility or photoperiod and/or thermosensitive male sterile lines addresses these problems. These lines responds to photoperiod, temperature or a combination of both which cause the plant to be fertile or sterile depending on the critical daylength or temperature [23]. With the two-line system using genetic male sterile, there is no need to develop a maintainer line and any fertile line can be used as a restorer. This greatly reduce the time and resources in making hybrid combination and parental seed production. Moreover, it broadens the available choices of restorers that can generate more combinations which in turn

Extensive studies suggest that genetic male sterile lines can be broadly classified into three categories; photoperiod genetic male sterile (PGMS), thermosensitive genetic male sterile (TGMS), and photoperiod and thermosensitive genetic male sterile (PTGMS) [15, 24, 25]. The first reported genetic male sterile came from spontaneous mutant in a japonica cultivar Nongken 58 discovered in Hubei China and were later called as Nongken 58S [2]. Further studies after its discovery revealed that the male sterility is regulated mainly by photoperiod and thus referred to as photoperiod genetic male sterile (PGMS). Nongken 58S showed complete pollen sterility when grown under long day conditions (>14 h), fertility was restored when

A thermosensitive type of male sterility was discovered in a spontaneous mutant AnnongS-1 (Ans-1) in 1997. The pollen remained sterile at both long and short day when exposed to 33°C and reverts back to fertile when the temperature reached 24°C [27]. Additional lines exhibiting thermosensitivity were also discovered in Zhu1S, Hengnong 1S and Guangzhang 63S where the fertility rates vary at different

The third classification of genetic male sterility affects both photoperiod and temperature. Pei'ai 64S is a line derived from the original male sterile mutant Nongken 58S with genetic backgrounds such as *indica* and *javanica* [31]. A study conducted on the response of Pei'ai 64S and another line 8902S showed fertility under long daylength (>14.5 h) and low temperature (24°C) or short daylength (10 h) and high temperature (28°C) conditions, but were consistently sterile at long

Numerous genetic studies concluded that genetic male sterility can be controlled by single, two genes or multiple genes depending on the genetic background and

*DOI: http://dx.doi.org/10.5772/intechopen.85191*

the sporophytic type CMS-WA [22].

**3. Discovery of genetic male sterile lines**

increases the probability of finding the best hybrids [4].

subjected to <10 h of light under controlled environment [26].

controlled temperatures regardless of daylength [28–30].

daylength (14 h) and high temperature conditions (28°C) [32].

*Genetics and Breeding System for Cytoplasmic and Genetic Male Sterility in Rice DOI: http://dx.doi.org/10.5772/intechopen.85191*

Extensive evaluation of lines and backcross breeding were employed to improve the lines and adapt to a particular environment [21]. Furthermore, new maintainers and restorers were developed from the original donors. A new CMS source was discovered in Dongxiang wild rice by continuous backcrossing to the *indica* variety Zongzao 35 [10]. In another study, a new source was found from interspecific cross of an *indica* with an African rice (*Oryza glaberrima Steud.*) that showed similarity to the sporophytic type CMS-WA [22].
