**2. Breeding component and system in hybrid rice development**

Rice is a strict self-pollinated crop; commercial exploitation of heterosis requires some parental specificity which could excludes manual emasculation. The invention of naturally occurred male sterility (MS) in rice thus played substantial role in realization of heterosis in rice. Following are the genetic tools as mentioned in various heads are required for development and commercialization of hybrid in rice:

#### **2.1 Male sterile system**

The male sterility (MS) in plants is the condition where the male reproductive organ, anthers, loses its ability to dehisce and produce viable pollen and thus encourages the allogamous nature of reproduction. This is crucial breeding tools to harness heterosis that exclude additional efforts of emasculation which is cumbersome process. In plants, male sterility is conditioned either by mitochondrial or nucleus genome or in associations. The male sterility in plant was first observed by Joseph Gottlieb Kolreuter in 1763 and later it was reported in >610 plant species. In rice, it was reported by Sampath and Mohanty [4] at ICAR-NRRI (formerly CRRI), Cuttack by studying the differences in male fertility in *indica/japonica* reciprocal crosses. The male sterility in plant is found to be determined by several biological as well as environmental factors. In rice, it is conditioned either by cytoplasmic genes in association with nuclear genes (CMS) or nuclear genes alone (GMS) which cause abnormal development in sporogenous tissue (either sporophytic or gametophytic tissue). The sporophytic male sterility is governed by genetic constitutions of sporogenous tissues like tapetal and meiocytes which creates improper nourishing to developing microspores and cause pollen abortion, whereas in gametophytic male sterility, microspore and pollen development get affected. Sporophytic male sterility is quite useful in hybrid rice breeding as it gets fertile in heterozygous state and encourages complete fertility in resulting hybrids. To date, several types of male sterile system, viz., cytoplasmic male sterile (CMS), environment sensitive male sterile (GMS), viz., thermo-sensitive genetic male sterility (TGMS), photo-sensitive genetic male sterility (PGMS) and reverse photo-sensitive genetic male sterility (rPGMS), etc. have been identified and substantially being utilized in hybrid development (**Table 1**).

#### **2.2 Diversity in male sterile system and their mechanism**

The CMS is a maternally hereditary trait instigated by improper communication between cytoplasmic and nuclear genome [5]. Gene(s)/genic block(s)-conditioned cytoplasmic male sterility is chimeric construct, which evolved due to rearrangement of the mitochondrial genome (**Figure 2**). In rice, several types of CMS have


**27**

**Table 1.**

*Cytoplasmic diversity in rice CMS.*

been identified and characterized, having diversified mechanism in MS expression. Wild abortive (WA-CMS), a sporophytic MS system, is widely utilized in hybrid development. It is found to be caused by a constitutive mitochondrial gene WA352c

Nuclease enzyme

regulator

*Hybrid Rice Research: Current Status and Prospects DOI: http://dx.doi.org/10.5772/intechopen.93668*

**ORF**

LX-CMS UK UK Luihui rice

Maxie-CMS UK UK MS mutant of

NX-CMS UK UK Selected from

Y-CMS UK UK Yegong (*indica*

PGMS *pms3* Noncoding RNA Nongken 58S,

P/TGMS *p/tms12–1* noncoding RNA Photoperiod

*Z*S1(loss in function)

*Note: "S" stands for sporophytic male sterility and "G" stands for gametophytic male sterility.*

rPGMS *csa* OsMST8 MYB transcript

protein

CW-CMS (G) *orf307* Mitochondrial

2. Environment-sensitive genetic male sterility (EGMS)

TGMS *tms5, RNase* 

**Protein Cytoplasm** 

**source**

(*indica*) cytoplasm

Maweizhan (*indica*) with Xieqingzao (*indica*)

F2 male sterile plants in the progeny of Wanhui 88 (*indica*) × Neihui 92–4 (*indica*) nucleus

landrace) cytoplasm

PGMS mutant of *japonica* cultivar Nongken 58

Spontaneous TGMS mutants of Annong S-1 and Zhu 1S

Carbon starved anther *(csa)* mutant of *japonica* cultivar 9522

*Oryza rufipogon* Griff.

and temperature sensitive genic male sterile (P/TGMS) derived from Nongken 58S

**Representative CMS-line**

Yue 4A

Maxie A

Neixiang 2A, Neixiang5A

Y Huanong A

IR24A, IR64A

7001S, N5088S

Pei'ai 64S

Guangzhan 63S5, Xinan S

9522S

**CMS group Associated** 


#### *Hybrid Rice Research: Current Status and Prospects DOI: http://dx.doi.org/10.5772/intechopen.93668*

*Recent Advances in Rice Research*

1. Cytoplasmic male sterile line a. *BT-CMS and their lineage*

b. *WA-CMS and their lineage*

**CMS group Associated** 

**ORF**

BT-CMS (G) *B-atp6-orf79* Membrane

HL-CMS (G) *atp6-orfH79* Membrane

WA-CMS (S) *rpl5-WA352* Membrane

LD-CMS (G) UK UK Lead Rice

Dian1-CMS (G) UK UK Yunnan

Kalinga-I-CMS (S) UK UK Kalinga-I

D-CMS (S) UK UK *Indica* rice

GA-CMS (S) UK UK Gambiaca

ID-CMS (S) UK UK Indonesia

K-CMS (S) UK UK K52(*japonica*)

protein

Membrane protein

CMS-RT102 (S) *rpl5-orf352* Membrane

*cox3*

CMS-RT98A (G) *orf113-atp4-*

DA-CMS (S) UK UK Dwarf abortive

**Protein Cytoplasm** 

protein

protein

protein

**source**

Chinsurah Boro II/ Taichong 65

(Burmese *indica* variety) × Fujisaka 5 (*japonica* variety)

high altitude landrace rice (*indica*) cytoplasm

Red-awned wild rice (*Oryza rufipogon*) cytoplasm

Wild abortive rice (*Oryza rufipogon*) cytoplasm

(*indica*) cytoplasm

Dissi D52/37

rice (*Oryza rufipogon*) cytoplasm

(*indica*) cytoplasm

paddy rice (*indica*) cytoplasm

cytoplasm

*Oryza rufipogon,* W1125

*Oryza rufipogon* Griff, W1109

**Representative CMS-line**

Liming A, Xu 9201A

Fujisaka 5A

Yongjing2A, Ning67A

Yuetai A, Luohong

Zhenshan97 A, V20A, IR58025A, CRMS31A, etc.

CRMS 32A

D-Shan A, D62A

Xieqingzao A

Gang 46A

II 32A, You1A

K-17A

RT102A

RT98A

3A4

**26**

#### **Table 1.**

*Cytoplasmic diversity in rice CMS.*

been identified and characterized, having diversified mechanism in MS expression. Wild abortive (WA-CMS), a sporophytic MS system, is widely utilized in hybrid development. It is found to be caused by a constitutive mitochondrial gene WA352c

#### **Figure 2.**

*Schematic presentation of rice CMS types, where WA stands for wild abortive, BT is for boro type, HL for Honglian, LD for lead rice, CW is for Chinese wild rice, RT102A and RT98A, respectively.*

located downstream of *rpl5* (comprised four mitochondrial genomic segments, *orf284, orf224*, *orf288*, and *cs4-cs6*) and encodes a putative protein (352-residue) with three transmembrane segments. The WA352c inhibits nuclear-encoded mitochondrial protein *COX11* (essential for the assembly of cytochrome c oxidase, TCA) and triggered premature tapetal programed cell death and pollen abortion [6]. In contrast, BT-CMS is a gametophytic MS reported in the Indian rice variety, Chinsurah Boro-II, in which pollen development get arrested at the tri-nucleate stage. The mitochondrial chimeric (dicistronic) gene *B-atp6-orf79* encodes a transmembrane protein, cytotoxic peptide*ORF79* [7], which accumulates preferentially in the microspore, was found to be responsible for male sterility. The *orf79* reside downstream to the *atp6* and interact with P61 and mitochondrial complex III and impair the activity of this complex which lead to dysfunctional energy metabolism and elevate oxidative stress and thus causing sterility. However, in HL-CMS, which is also a gametophytic MS system, pollen development gets arrested at di-nucleate stage. A chimeric aberrant transcript of the mitochondrial gene*atp6-orfH79*, located downstream of *atp6*is confirmed as candidate gene of this MS. Transcript of *orfH79* gene preferentially accumulates in mitochondria which interacts with P61 (a subunit of ETC complex III) and impairs mitochondrial function [8] and leads to MS. The MS in CW-CMS is conditioned by mitochondrial *orf307*, which causes anther-specific mitochondrial retrograde regulation for nuclear gene expression. It is a gametophytic MS in which pollen grain appears normal but unable to germinate.

#### **2.3 Genetic male sterility (GMS)**

The GMS in rice is conditioned generally by recessive nuclear genes and exert showing normal Mendelian inheritance. Owing to difficulties in their maintenance (occurrence of only 50% sterility in F1), GMS could not be part of rice hybrid breeding program. Some GMS lines has shown threshold nature in MS expression

**29**

*Hybrid Rice Research: Current Status and Prospects DOI: http://dx.doi.org/10.5772/intechopen.93668*

two-line hybrids in tropics and subtropics [9].

**2.4 Transgenic cytoplasmic male sterility**

gene *tms5* through CRISPR/Cas9.

**2.5 Genetics of fertility restorer gene**

where male sterility occurs in specific environmental regime (high temperature and long day length); hence called environment sensitive genetic male sterile (EGMS). The GMS line shows male sterility at elevated temperature, that is, >30°C is called temperature sensitive male sterility (TGMS) whereas male sterility in long day length, that is, >13.5 h is called photoperiod-sensitive genetic male sterility (PGMS). The male sterility in EGMS line is found to be revert into male fertile in favorable temperature (<30°C) and day length (<12.5 h) which provide its unique opportunity to be utilized in hybrid rice breeding program. The rice lines exert MS impression under long photoperiod and elevated temperature are referred as P/ TGMS, for example, Pei'ai 64S. The EGMS lines, PGMS-Nongken 58S (NK58S) and TGMS-Annong S-1 and Zhu1S or derivatives are utilized extensively in majority (>95%) of the two-line hybrid program. Among, derivatives of NK58S are exerts either P/TGMS or even TGMS (e.g., Guangzhan 63S), the mechanism underlying to such dramatic changes yet to be revealed. Recently, a novel type of EGMS (*csa*carban starved anther mutant) in rice called rPGMS (reverse PGMS). These lines expresses MS under short photoperiod (<12.5 h) and revert to normal fertile when exposed to long days (>13.5 h). This is found to be suitable for seed production of

The genetically engineered male sterile line M2BSin rice is developed by transformation of *indica* rice maintainer M2B with partial-lengthHcPDIL5-2a (Hibiscus cannabinus protein disulfide isomerase-like) genetic construct. Male fertility in this CMS is reported to be arrested due to tapetum degeneration which leads pollen abortion. The genetic analysis revealed this MS a maternally inherited inability as of CMS. Besides, by combining cysteine-protease gene (BnCysP1) of Brassica napus with rice anther-specific P12 promoter (promoter region of *Os12bglu38* gene), a transgenic MS system was successfully created which is restored by transgenic rice plants carrying BnCysP1Si silencing system [10]. Zhou and co-workers [11] could develop 11 "transgene clean" TGMS lines by editing most widely utilized TGMS

The rice CMS is found to be restored by nuclear genome, that is, mono or oligo nuclear loci called restorer gene. In rice, a total of 10 *Rf* genes (*Rf1a*, *Rf1b*, *Rf2*, *Rf3*, *Rf4*, *Rf5*, *Rf6* and *Rf17, Rf98 and Rf102)* have been identified, of those seven (*Rf1a, Rf1b, Rf2, Rf4, Rf5*, *Rf17*, and *Rf98*) are characterized. All *Rf* genes are found to be dominant in nature (except *Rf17,* restores fertility in CW-CMS), which can restore male fertility in heterozygous state. Restorer genes are very specific to male sterile genome in the mechanism of fertility restoration. Genes *Rf1a* and *Rf1b* (Chr.-10) encode pentatricopeptide-repeat (PPR)-containing proteins and have functional affinity of fertility restoration in *BT-CMS*; RF1A promotes endonucleolytic cleavage of the *atp6-orf79* mRNA andRF1B promotes degradation of *atp6-orf79* mRNA [7] and revert the male sterility into fertility. Whereas, HL-CMS is restored either by *Rf5 or Rf6* gene, these genes can produce 50% normal pollen grains in F1 plants individually; however, both genes in complementation could restore more than 80% spikelets' fertility in hybrids. The *Rf5* encodes a PPR family protein PPR791 and which bind with GRP162 (glycine rich protein) and *atp6-orfH79* transcripts and makes a RFC (restoration of fertility complex). The RFC cleave the aberrant transcript of *atp6-orfH79*at 1169 nucleotides position [12]. The*Rf6* gene encodes a novel PPR family protein (duplicate PPR motif 3–5)

*Hybrid Rice Research: Current Status and Prospects DOI: http://dx.doi.org/10.5772/intechopen.93668*

*Recent Advances in Rice Research*

**28**

**2.3 Genetic male sterility (GMS)**

**Figure 2.**

*Schematic presentation of rice CMS types, where WA stands for wild abortive, BT is for boro type, HL for* 

located downstream of *rpl5* (comprised four mitochondrial genomic segments, *orf284, orf224*, *orf288*, and *cs4-cs6*) and encodes a putative protein (352-residue) with three transmembrane segments. The WA352c inhibits nuclear-encoded mitochondrial protein *COX11* (essential for the assembly of cytochrome c oxidase, TCA) and triggered premature tapetal programed cell death and pollen abortion [6]. In contrast, BT-CMS is a gametophytic MS reported in the Indian rice variety, Chinsurah Boro-II, in which pollen development get arrested at the tri-nucleate stage. The mitochondrial chimeric (dicistronic) gene *B-atp6-orf79* encodes a transmembrane protein, cytotoxic peptide*ORF79* [7], which accumulates preferentially in the microspore, was found to be responsible for male sterility. The *orf79* reside downstream to the *atp6* and interact with P61 and mitochondrial complex III and impair the activity of this complex which lead to dysfunctional energy metabolism and elevate oxidative stress and thus causing sterility. However, in HL-CMS, which is also a gametophytic MS system, pollen development gets arrested at di-nucleate stage. A chimeric aberrant transcript of the mitochondrial gene*atp6-orfH79*, located downstream of *atp6*is confirmed as candidate gene of this MS. Transcript of *orfH79* gene preferentially accumulates in mitochondria which interacts with P61 (a subunit of ETC complex III) and impairs mitochondrial function [8] and leads to MS. The MS in CW-CMS is conditioned by mitochondrial *orf307*, which causes anther-specific mitochondrial retrograde regulation for nuclear gene expression. It is a gametophytic MS in which pollen grain appears normal but unable to germinate.

The GMS in rice is conditioned generally by recessive nuclear genes and exert showing normal Mendelian inheritance. Owing to difficulties in their maintenance (occurrence of only 50% sterility in F1), GMS could not be part of rice hybrid breeding program. Some GMS lines has shown threshold nature in MS expression

*Honglian, LD for lead rice, CW is for Chinese wild rice, RT102A and RT98A, respectively.*

where male sterility occurs in specific environmental regime (high temperature and long day length); hence called environment sensitive genetic male sterile (EGMS). The GMS line shows male sterility at elevated temperature, that is, >30°C is called temperature sensitive male sterility (TGMS) whereas male sterility in long day length, that is, >13.5 h is called photoperiod-sensitive genetic male sterility (PGMS). The male sterility in EGMS line is found to be revert into male fertile in favorable temperature (<30°C) and day length (<12.5 h) which provide its unique opportunity to be utilized in hybrid rice breeding program. The rice lines exert MS impression under long photoperiod and elevated temperature are referred as P/ TGMS, for example, Pei'ai 64S. The EGMS lines, PGMS-Nongken 58S (NK58S) and TGMS-Annong S-1 and Zhu1S or derivatives are utilized extensively in majority (>95%) of the two-line hybrid program. Among, derivatives of NK58S are exerts either P/TGMS or even TGMS (e.g., Guangzhan 63S), the mechanism underlying to such dramatic changes yet to be revealed. Recently, a novel type of EGMS (*csa*carban starved anther mutant) in rice called rPGMS (reverse PGMS). These lines expresses MS under short photoperiod (<12.5 h) and revert to normal fertile when exposed to long days (>13.5 h). This is found to be suitable for seed production of two-line hybrids in tropics and subtropics [9].

#### **2.4 Transgenic cytoplasmic male sterility**

The genetically engineered male sterile line M2BSin rice is developed by transformation of *indica* rice maintainer M2B with partial-lengthHcPDIL5-2a (Hibiscus cannabinus protein disulfide isomerase-like) genetic construct. Male fertility in this CMS is reported to be arrested due to tapetum degeneration which leads pollen abortion. The genetic analysis revealed this MS a maternally inherited inability as of CMS. Besides, by combining cysteine-protease gene (BnCysP1) of Brassica napus with rice anther-specific P12 promoter (promoter region of *Os12bglu38* gene), a transgenic MS system was successfully created which is restored by transgenic rice plants carrying BnCysP1Si silencing system [10]. Zhou and co-workers [11] could develop 11 "transgene clean" TGMS lines by editing most widely utilized TGMS gene *tms5* through CRISPR/Cas9.

#### **2.5 Genetics of fertility restorer gene**

The rice CMS is found to be restored by nuclear genome, that is, mono or oligo nuclear loci called restorer gene. In rice, a total of 10 *Rf* genes (*Rf1a*, *Rf1b*, *Rf2*, *Rf3*, *Rf4*, *Rf5*, *Rf6* and *Rf17, Rf98 and Rf102)* have been identified, of those seven (*Rf1a, Rf1b, Rf2, Rf4, Rf5*, *Rf17*, and *Rf98*) are characterized. All *Rf* genes are found to be dominant in nature (except *Rf17,* restores fertility in CW-CMS), which can restore male fertility in heterozygous state. Restorer genes are very specific to male sterile genome in the mechanism of fertility restoration. Genes *Rf1a* and *Rf1b* (Chr.-10) encode pentatricopeptide-repeat (PPR)-containing proteins and have functional affinity of fertility restoration in *BT-CMS*; RF1A promotes endonucleolytic cleavage of the *atp6-orf79* mRNA andRF1B promotes degradation of *atp6-orf79* mRNA [7] and revert the male sterility into fertility. Whereas, HL-CMS is restored either by *Rf5 or Rf6* gene, these genes can produce 50% normal pollen grains in F1 plants individually; however, both genes in complementation could restore more than 80% spikelets' fertility in hybrids. The *Rf5* encodes a PPR family protein PPR791 and which bind with GRP162 (glycine rich protein) and *atp6-orfH79* transcripts and makes a RFC (restoration of fertility complex). The RFC cleave the aberrant transcript of *atp6-orfH79*at 1169 nucleotides position [12]. The*Rf6* gene encodes a novel PPR family protein (duplicate PPR motif 3–5)


**Table 2.** *Restorer genes in rice plants.*

which in association with hexokinase (*osHXK6*) targets mitochondria and process defective transcript of *atp6-orfH79* at 1238 nucleotide position. Thus, PPR protein family cause editing of aberrant transcript, inhibit their translation, and at the end, fertility restoration. Besides, male fertility in WA-CMS is found to be counteracted by *Rf3* and *Rf4* genes (chrom.-1 and 10, respectively). The genes *Rf3* and *Rf4* encode a pentatricopeptide protein (PPR) where RF4 cleave the abnormal *WA352* transcript and RF3 suppress translation of *WA352* into polypeptide and helps in restoring fertility in WA-CMS. Fertility in LD-CMS is reported to be restored by either *Rf1* or *Rf2.* The *Rf2* gene encodes a glycine-rich protein in mitochondrial; replacement of isoleucine by threonine at amino acid 78 of the *RF2* protein causes functional loss of the *rf2* allele. Moreover, CW-CMS is reported to be restored by a single recessive gene (*Rf17)* which is a retrograde-regulated male sterility (*rms*) gene (**Table 2**) [20].

#### **2.6 Breeding system**

Commercial hybrid seed production in rice where natural out-crossing (ranged only 0.3–3.0%) is very low, cumbersome, and an expansive task. To be practical and readily adoptable, it requires some specific parental requirements and agro-management practices. Invention of male sterile lines thus provided unique opportunity to start with the technology in rice. Based on mechanism of male sterility, threshold nature in male sterility expression and number of parental lines used, three types of hybrid seed production system namely three-line system (involving three parents, A, B, and R), two-line system (two parents, A and R), and one-line system (apomictic-based) exist. Among them, CGMS-based three-line system is more suitable,

**31**

*Hybrid Rice Research: Current Status and Prospects DOI: http://dx.doi.org/10.5772/intechopen.93668*

rice varietal development and seed production.

*2.6.1 CGMS system*

and heat at flowering stage.

gain in hybrids.

*2.6.2 EGMS system*

below CSP of photoperiod length, <12.5 h.).

hence widely utilized (>90% of world's hybrids developed utilizing this) in hybrid

This system involves three parents such as male sterile line (A-line, cytoplasmic male sterile), B-line (maintainer), and R (restorer) lines and two steps in seed production, that is, CMS multiplication and hybrid seed production under strict isolation (spatial or temporal or physical barrier). Male sterile line (A-line), because of their eliminated manual emasculation needs, served as seed parent and facilitates large-scale seed production. A suitable CMS line to be utilized as seed parent should have complete and stable male sterility, substantial seed producibility, wide compatibility, and good combining ability with minimum CMS load. The wealthy panicle and narrow semi-erect leaf configuration in seed parent has additional impact, assures more seed production. In Indian perspective, hybrid seed production is a major dilemma, generally keen to *Rabi* season, hence, CMS lines should have substantial cold tolerance at seedling stage

The maintainer (B-line), on the other hand, is an isogenic to the CMS line (differs only for fertility/sterility) in their genetic constitution, able to produce functional pollen and maintain the sterility in male sterile line/seed parent. The maintainer line can maintain 100% male sterility in seed parent thus utilized to

In contrast, restorer line can restore male fertility in F1s produced on male sterile parent, thus utilized as pollen parent in hybrid seed production. A good restorer should have substantial genetic distance with seed parent which is prerequisite and major determinant of the extent of heterosis in hybrids (more genetic distance more heterosis and *vice-versa*). Restorer is the major contributor of heterosis in three-line hybrids, hence, should have good combining, strong fertility restoration ability (dominant *Rf* gene(s) responsible for fertility restoration in CMS). Besides, restorer line with ideal plant type, acceptable grain quality parameters, substantial source-sink balance, heavy pollen load, and broad spectrum of resistance/tolerance against multiple biotic/abiotic stresses is imperative in maximization of genetic

This system is a simple and more efficient hybrid breeding system in rice, involves only two parents, that is, A and R line in seed production, thus, referred as two-line system. This is a threshold of genetic male sterility (EGMS)-based hybrid rice breeding system, where male sterility is conditioned in specific environmental regimes such as long photoperiod (>13.5 h day length) and at elevated temperature (>30°C). In this system, male sterile parents are to be maintained by selfing under favorable conditions (below critical sterility point, i.e., <30°C temperature and at

Two-line hybrid seed production system is an easy and effective alternative to CMS and has specific advantages as it requires only one step for seed production. In this system, any good combiner genotype irrespective of their fertility restoration ability can be utilized as a pollen parent. EGMS system is normal and does not exert any ill effect in the growth and development of carrier plant, and thus, exploits comparatively higher extent of heterosis (up to 5–10%) in F1 than the CGMS-based three-line system. The EGMS traits are governed by major genes, thus are easily

perpetuate CMS with their inherent male sterile ability.

hence widely utilized (>90% of world's hybrids developed utilizing this) in hybrid rice varietal development and seed production.
