**4. Wild species**

The genus Oryza contains tweny valid species, two of which are cultivated, namely *Oryza sativa* and *Oryza glaberrima.* There are nine diploid species among the remaining 18 species (**Table 2**). Six of them are tetraploid. Some of the wild species utilized in breeding programme are *Oryza perennis* - Co 31 GEB 24 x *O. perennis* [8–10].

#### **5. Breeding component and system in hybrid rice development**

For breeding technique, there are three approaches (1) the three-line method also known as CMS (cytoplasmic male sterility) system (2) the two-line method also known as the PTGMS (photo/temperature sensitive genic male sterility) system and (3) the one-line method, also known as the apomixis system. Inter-varietal hybrids, Inter-sub-specific hybrids and inter-specific or intergeneric hybrids are three ways to increase the degree of heterosis (**Table 3**).

#### **5.1 Two-line hybrid rice**

The two-line hybrid rice research began in China and was successfully scaled up in 1995. The thermo-sensitive male sterile lines (TGMS) lines are those whose sterility expression is regulated by temperature, whereas photoperiod-sensitive male sterile (PGMS) lines are those whose expression is controlled by day-length duration. Backcrossing has successfully transferred the PGMS trait to many *Indica* and *Japonica* rice cultivars in China. In China, rice hybrids produced by this male sterile system are being tested in multiple locations. The degree of heterosis in two-line hybrid rice is close to that of three-line hybrid rice, but the technique methods is different. Unlike three-line hybrids, the male parent of two-line hybrid is not limited by restorer genes, allowing us to use both good restorer lines with high combining potential and good traditional varieties without restorer genes as male


**Table 2.**

*Wild species of Rice.*

parents. Since restorer genes are not limited, there's a better chance of breeding elite hybrids [12]. The developed PTGMS lines such as PA64S, GZ63S, Zhun S, etc. have many advantages for hybrid combinations, such as larger freedom for crossing, higher yielding, better quality and diseases resistance. The yield of improved two-line hybrid rice combinations is usually higher than of three-line hybrids used as controls. Meanwhile, seed processing and cultivation techniques for two-line hybrids have advanced to the point that they can be used in commercial production. Breeding of elite restorer lines is the key for matching heterotic combinations [13] (**Table 4**).

#### **5.2 Three-line system hybrid rice**

#### *5.2.1 Identification and utilization of cytoplasm male sterility*

The role of rice cytoplasm in male sterility was first discovered in 1954 [16]. They studied cytoplasmic differences among rice varieties in 1965 and formed a male sterile line for the first time by transferring the nuclear genotype of rice cultivar Fujisaka [17]. However, due to its instability, poor plant form and photoperiod sensitivity, this cytoplasm male sterility (CMS) line could not be used to breed rice. Yuan Long Ping proposed the concept of using heterosis in rice in 1964, and for the




*Source: [11].*

#### **Table 3.**

*List of hybrid Rice released/notified in India during 2010–2017.*

first time in China, hybrid rice research was started. The discovery of WA, a nationwide cooperative program was immediately established to extensively testcross with the WA and screen for its maintainers and restorers. Soon in 1972, the first group of CMS lines such as Erjiunan 1A, Zhenshan 97A and V20A were developed all using WA as the donor of male sterile genes and all using successive backcrossing method. In 1973, the first group of restorer lines such as Taiyin 1, IR24 and IR661 were screened using direct test crossing system. Nanyou 2 and Nanyou 3 hybrids




*Grain: LS: Long Slender, MB: Medium Bold, MS: Medium Slender, SB: Short Bold, MB: Medium Bold.*

#### **Table 4.**

*Rice varieties developed by ICAR-NRRI, Cuttack during 2010–2019.*

with high heterosis were published in 1974 [18–20]. In another word, the discovery of WA led to successful breakthrough in hybrid rice production, resulting in the establishment of three-line hybrid rice system. As a result, China became the first country in the world to commercialize hybrid rice for food production. For commercial rice hybrids processing, a three-line hybrid system with the CMS line (A), maintainer line (B) and restorer line (R) is used. The A line cannot produce viable pollen due to the interaction between cytoplasmic and nuclear genes, so called

cytoplasmic male sterile, which anthers are pale or white and shriveled. The A line is also known as the CMS line and the seed parent because it is used as a female parent for hybrid seed development. Since the CMS line is male sterile, it cannot replicate itself and requires the assistance of a maintainer. The B line is the maintainer line, and its morphology is very similar to that of its CMS line, with the exception of its reproductive feature. However, the B line has viable pollen grains and normal seed setting, it may pollinate the A line, resulting in male sterile F1 plants. In this way, the male sterility of the A line is maintained, and the A line can be reproduced for further use or commercial purposes. Similarly, the R line will pollinate the A line because it has viable pollen grains and normal seed setting. Unlike the pollination with the B line, the F1 plants from the pollination with R line are extremely fertile, or the male sterility of the A line is restored into fertility in their progeny by R line. As a result, the R line is often referred to as the pollen parent or restoring line [21–25].
