**7. Breeding of TGMS Lines**

Procedures for breeding TGMS lines are similar to conventional breeding procedures, with one major difference. The trait to be selected is male sterility, which requires a particular set of conditions for it's expression and another set of conditions for multiplication of selected segregants. Sterile plants are selected under appropriate conditions in F2 generation. Such plants are ratooned and grown under fertility inducing conditions to obtain seed of the selected segregants. Since TGMS is a recessive character, and if it is controlled by a single gene in selected segregants, there will be no further segregation for sterility/fertility in F3 and subsequent generations, though there may be segregation for other plant characters. Thus F3 generation onwards, the selected segregants can be grown in fertility inducing conditions for selection and forwarding the generations. By F6 generation, stabilized elite TMGS lines can be developed.

#### **8. Breeding procedures for TGMS Lines**

#### **8.1 Germplasm search**

Make a detailed and systematic study on germplasm or any stabilized breeding material and look out for spontaneous sterile mutants which may revert to fertility under low temperature. Robin *et al*., [62] developed the new TGMS line (TNAU 60S) and was identified as spontaneous mutant from the rice variety PMK 3 with desirable floral characteristics and stable sterility. This TGMS line has the duration of 125 days with semi dwarf plant type. The panicle exertion percentage is 76.9% with wide angle of glume opening which makes the line with higher out crossing potential highly amenable for commercial exploitation. The grain quality of the TGMS line is highly preferable. TNAU 60 S has been used in hybridization and many heterotic hybrids were developed. This line was registered with NPBGR for its unique TGMS trait as IC 0622805 and INGRI 17028.

#### **8.2 Pedigree breeding**

Agronomically adopted line or variety will be crossed with TGMS donor and F1 will be studied under normal Environments and selfed seeds of F1 will be raised under sterility inducing Environment. From this F2 population Sterile plants will be are selected under high temperature *ie* sterility inducing Environment. Such plants are ratooned and grown under fertility inducing conditions to obtain seed of the selected segregants. Since EGMS is a recessive character, and if it is controlled by a single gene in selected segregants, there will be no further segregation for sterility/fertility in F3 and subsequent generations, though there may be segregation for other plant characters. Thus F3 generation onwards, the selected segregants can be grown in fertility inducing conditions for selection and forwarding the generations. By F6 generation, stabilized elite TMGS lines can be developed. Kavithamani et al. [63] developed the many new tgms lines by crossing CO 49 an agronomically adopted variety with TS 29 (promising TGMS line) as a donor line. The lines shows superiority over their parents in sterility with good floral traits and grain yield. The details were given in **Figure 1**.

One of the line were characterized by [64]. TNAU 135S (TS 29/CO 49) with a long duration line showed 100% sterility during January month sowing at Department of Rice, Coimbatore and its complete sterility is under maximum temperature of 31.2°C, and minimum temperature of 21. 0 C and critical stage for expression of pollen sterility is 85–104 days. Other lines TNAU 137S developed from TNAU TNAU4S-1-2 /CB 06–564 with CSP of 20.3 also showed complete sterility whereas another line with same female parent TNAU 4S with BPT 5204 showed partial sterility indicated that it requires different month of sowing.

#### **8.3 Molecular tagging of TGMS gene and development of new lines**

The TGMS parental line, TS 29 has a stable sterile and fertile phases with substantially low critical temperature. Inheritance studies of the F2 population revealed that the thermo-sensitive genic male sterility in TS 29 was under the control of single recessive gene. Molecular tagging of TGMS genes in the F2 mapping population was done by using SSR markers. Out of 50 primer pairs (putatively linked to the six reported TGMS genes in rice) assayed for studying polymorphism, 19 primer pairs produced polymorphic alleles between the parents. The SSR markers revealed 38 percentage of polymorphism between TS 29 and CO(R) 49, the recipient fine grain parent. The identified 19 primer pairs were used for bulked segregant analysis A total of 400 F3 progenies were raised during summer (fertility

*Progress and Prospects of Two Line Rice Breeding in India DOI: http://dx.doi.org/10.5772/intechopen.99545*

limiting season). The DNA of F2 plant which contributed the sterile/ fertile F3 plants were identified and bulked. The study identified one SSR marker, RM 3476 which co-segregated with the phenotypic observations recorded under the field condition. The marker, RM 3476 has already been identified as located adjoining *tms 6* gene in chromosome 5 of rice. Population advancement for fixing stable segregants with TGMS trait and diversifying the resistance through other crosses and MAS are in progress. However the stability and heterotic potential of TGMS segregant lines were assessed in F4 generation by crossing with tester parents. The F1s were evaluated along with check hybrids few promising hybrids *viz.*, TNAU 61 S/DE 2, TNAU 31S/ JGL 385 were identified as superior in grain yield and grain quality. Robin *et al*., [60].

#### **8.4 Mutation breeding**

Any breeding materials can be mutated and the progenies will be screened for the presence of TGMS gene. Strict observations are to be made in M2 generation planted under high temperature region, as the trait is governed by a recessive gene. To improve the floral traits favoring out crossing two stable TGMS lines *viz.*, TS 6 and CBTS 0282 were subjected to gamma rays (300 and 350 Gy). In the M2 generation, a total of 469 progeny rows with 1,28, 975 plants of CBTS 0282 and 854 progeny rows with 1,28,100 of TS 6 were raised. 361 sterile plants with good stigma exertion and wide angle of glume opening were selected and stubble planted at Hybrid Rice Evaluation Centre, Gudalur, a low temperature region for inducing pollen fertility and further seed increase. The M3 and M4 generations of the selected plants with desirable floral traits were evaluated at high temperature conditions at Coimbatore and seed increase was done at Gudalur. Finally a total of 11 TGMS lines were developed with good floral traits *viz.*, better stigma exertion percentage, wider angle of glume opening and better panicle exertion than the control were identified and are being utilized for two line hybrid rice development [65]. Out of these 11 lines two lines TNAU 84S (TS 29 150Gy) and TNAU 139S(TS 29 100 Gy-3) were characterized by [64] and found that TNAU 139S was completely sterile in all five staggering during January sowing but TNAU 84S did not showed complete pollen fertility but it showed complete spikelet sterility. TNAU 139S recorded CSP of 20.3. Apart from using already available TGMS lines released varieties were also used for developing new TGMS lines.

Mutation studies were further followed with released rice varieties by [66]. Two rice varieties viz., ADT 39 and CR 1009 were utilized to generate genetic variability by exposing them to gamma rays at 50, 100, 150, 200, 250 and 300 Gy. The main focus of this study is to identify TGMS mutants which could help in hybrid breeding programme. Chlorophyll mutants were observed in both the varieties in M2 generation. The male sterile plants were identified in M2 generation under high temperature condition (Coimbatore) and the reverted lines in the low temperature region (Gudalur) were planted again in the high temperature condition to confirm their TGMS nature. All the plants expressed complete sterility. Seven plants (comprising five plants from ADT 39 and two plants from CR 1009) isolated from M3 generation recorded 100 percent pollen and spikelet sterility under high temperature condition and more than 60 percent spikelet fertility under low temperature condition. These lines were further advanced for attaining homozygosity and out of seven lines one of the promising line TNAU 100S was Isolated from the ADT 39 100 Gy. The line was characterized and found that 72–91 days were critical stages for expression of pollen sterility and CSP was 20.5°C [64]. This line also showed complete sterility

with wider sterility period. This line is having good grain quality *ie* medium slender grain type which is highly preferred by south Indian people can be exploited for commercial two line hybrid development.

#### **8.5 Backcross programme**

Transferring the genes from already available sources to elite genotypes or lines with high combining ability. Tanee et al., [67] utilized this metod for developing new TGMS lines. To transfer *tgms* gene(s) controlling TGMS to Thai rice cultivars by backcross breeding method, Thai rice cultivars ChaiNat 1, PathumThani 1, and Suphan Buri 1 were used as recurrent parents and a male sterile line was used as a donor parent. The BC2F2 lines were developed from backcrossing and selfing. An individual plants were evaluated for tmsX gene by conventional breeding and 28 plants were selected from the total plants 78. A total of 18 SSR markers covering the 12 rice chromosomes were employed to select the outstanding genetic background for backcrossing in order to maintain genetic background of the recurrent parent. Selected 60 plants were screened for tmsX gene by phenotyping, subsequently 32 selected plants were screened by added more 18 SSR markers for genetic background in BC2F1 generation. In order to increase the recurrent parent genetic background, MAS was applied at BC2F1 generation by using more number of loci for providing opportunity to find individual plants with the highest genetic background of the recurrent parent. Two plants were isolated from BC2F1 generation and were found to carry 97.22% genetic background. These plants had genetic background higher than BC2F1 generation. Selfed seeds from selected BC2F1 plants were planted at a temperature higher than 30°C and phenotypic selection was employed at flowering stage for selection of the sterile plants. In this way they successfully introgressed the *tgms* gene into Thai rice cultivars.

## **9. Characterization of the TGMS lines**

To study the critical fertility (CFT) and critical sterility (CST) point for TS 29, staggered sowing of seeds in weekly interval was taken up in large cement pots. Pollen fertility was observed. Fertility variation in comparison with maximum temperature prevailed during that month was compared. Complete sterility was observed at a temperature of more than 32/19°C and it was fertile below this temperature. But occasionally sterility was observed above and below this temperature that may be due to combined influence of the other weather parameters. The results of correlation analysis between pollen sterility and weather factors revealed that maximum and mean temperature were the primary factors influencing fertility transition. In this result the negative association was observed between pollen sterility and relative humidity. During the sterile phase relative humidity was low (< 85 per cent) and during fertile phase relative humidity was high (> 90 per cent). Minimum temperature was also observed a significant association with pollen sterility in the latter phase of panicle developments. Sunshine hours had lower level of influence over pollen sterility. Negative significant association was observed between relative humidity and pollen sterility percentage (**Figures 2–4**) [60].

A study was carried out an experiment at the Paddy Breeding Station, TNAU, Coimbatore. The materials comprised, 60 suspected TGMS lines from different populations viz., for screening the sterility /fertility expression. All the population of TGMS lines initiated panicle development during April when the maximum/

*Progress and Prospects of Two Line Rice Breeding in India DOI: http://dx.doi.org/10.5772/intechopen.99545*

#### **Figure 2.**

*Fertility behavior of TS 29 for maximum temperature.*

**Figure 3.**

*Fertility behavior of TS 29 for minimum temperature.*

**Figure 4.** *Fertility behavior of TS 29 for mean temperature.*

minimum temperature (day/night) was 30.5–37.8 0 C/22.0–26.4°C. The suspected TGMS lines were evaluated for pollen fertility by using 1% Iodine Potassium Iodide (I-KI) solution. Pollen grains from three randomly chosen fields were evaluated and pollen fertility was expressed in percentage. Five panicles per plant were evaluated for spikelet fertility. Sterile plants identified from promising TGMS lines were ratooned for self multiplication of seeds to confirming fertility transformation during kharif. Pollen fertility/spikelet fertility observation were recorded for ratooned plants. Seeds were collected from each ratooned promising TGMS lines and raised for evaluation during rabi. Pollen/spikelet fertility was assessed for all TGMS lines. Promising TGMS lines identified during summer. Out of 60 population evaluated for TGMS expression, 175 sterile plants were identified based on pollen/spikelet sterility. The sterile plants consisted of 27 F4'S, 40 Fs's, 20 DH's and 88 GD No′s. The results showed that 25 lines found promising for stable TGMS expression along with good floral traits. Among the TGMS lines CBTS 0280, CBTS 0283, CBDHTS 025, GD 98014, GD 98028 had early flowering. All the twenty five promising TGMS lines exhibited 100 per cent pollen/spikelet sterility during summer season when the maximum, minimum temperature was 30.5–37.8/22.0–26.4 (day/night). The TGMS lines viz., CBTS 0268 and CBTS 0272 are found to be possessing long slender grain, purple tip, well exerted purple stigma. The TGMS line viz., CBTS 0252 and CBTS 0254 were developed from Indica/Japonica crosses which showed 100% pollen sterility, medium slander grain with purple stigma. The results revealed that the TGMS line viz., CBTS 0252 and CBTS 0254 could be useful to produce two line hybrids with high heterosis for yield with good plant type [68].

Newly developed 66 tgms lines were screened under sterility favoring environment for tgms gene expression during summer and sterility limiting environment during winter at Paddy Breeding Station, Coimbatore for the past five years. Out of 66 TGMS lines, 15 lines showed stable performance and seven TGMS lines *viz*., COTGMS 02, COTGMS 07, COTGMS 10, COTGMS 11, COTGMS 12, COTGMS 13 and COTGMS 15 were completely pollen sterile through out the summer period. It shows that, these lines are having wider sterility expression period. These lines also recorded very good floral traits *viz.,* higher pollen sterility per cent, panicle and stigma exsertion per cent, wider glume opening favorable for enhancing out-crossing rate and seed set percentage during seed production [69].

A total of 21 TGMS lines which showed complete sterility were raised during kharif for seed multiplication in the fertile phase [70]. Among the 21 TGMS lines, 12 TGMS lines viz., TNAU 9S, TNAU 14S, TNAU 15S, TNAU 28S, TNAU 30S, TNAU 32S, TNAU 63S, TNAU 64S, TNAU 67S, TNAU 69S, CBTS 0282-27-1 and TS 6–182-1 were found to be highly fertile and uniform in plant and grain type. Utilizing these lines Crossing block with 7 TGMS lines and 120 male parents were raised for developing new two line hybrid combinations and 23 hybrids were synthesized. All these hybrids were evaluated with already existing hybrids and promising hybrids were forwarded. A total of 9 two line rice hybrids were raised in the advanced yield trial to assess the yield performance along with the check varieties / hybrids viz., CO 48, CORH 3, CORH 2 and ADTRH 1. Six hybrids viz., TNTRH 1, TNTRH 2, TNTRH 5, TNTRH 8, TNTRH 10 and TNTRH 12 performed well over the checks for which mini seed production has been taken up for further evaluation. First time in India these two line hybrids were developed and evaluated under yield trial. The hybrid combination viz., TNTRH 5 has been nominated for Initial hybrid rice trial (IHRT-Medium) during Kharif 2007. This hybrid recorded 6893 kg/ha of grain yield. The promising medium duration hybrid TNTRH 19 recorded the grain yield of 8210 kg/ ha which is 28.12% increased yield over CO (R) 49 and was tested under MLT 2010 hybrid Rice – Medium Trial.

Salgotra et al. [71] characterized eight TGMS lines, DDR 1S, DDR 18S, DDR 19S, DDR 20S, DDR 23S, DDR 27S, DRR 28S and DDR 29, showed complete sterility at low altitude and satisfactory seed-set percentage at high altitude. Characterization of floral traits and sterility-sensitive stage were determined by the tracking method. At low altitude, with an average air temperature of 35.4°C, TGMS lines DRR 19S, DRR 20S and DRR 29S displayed a sterility-sensitive stage at 21 days prior to normal heading. For complete sterility the TGMS line DRR 1S requires a temperature of 36.6°C at 17 days prior to normal heading. The temperature for complete sterility ranged from 33.9°C to 35.8°C at low altitude in the remaining seven lines. A significant positive correlation with opening duration of lemma and palea and with size of stigma and angle of opened lemma and palea.

## **9.1 Out crossing potential of TGMS lines**

The newly developed TGMS Lines are to be assessed for its outcrossing potential then only it can be successfully exploited for hybrid breeding programme. [72] studied the outcrossing potential of TGMS lines. In this study average style length of the TGMS lines was 1.87 mm. Maximum style length was recorded in TNAU 18 S with 2.01 mm while TS-29–150 GY and TNAU 60 S had the styles with 1.85 mm and 1.75 mm, respectively. TNAU 18 S had the maximum spikelet opening angle of 23.010 and the TS-29–150 GY and TNAU 60 S had the angles of 20.23<sup>0</sup> and 18.540, respectively. TNAU 60 S involved in five cross combination and the minimum of 60.8 cm and the maximum of 74.4 cm plant height was obtained. Similarly TS-29–150 GY had the minimum height of 62.2 cm and the maximum plant height of 66.80 cm involving in two cross combinations. It was noted that the tillering and flowering of TGMS lines were prolonged even after the completion of flowering in male parents. TNAU 18 S showed the 92.8 per cent with highest panicle exertion rate, TS 29 150 GY had the medium value of 66.6 per cent while TNAU 60 S had the lowest of 51.8 per cent. This parameter did not show significant difference among the lines tested. The degree of spikelet opening angle and the duration of panicle opening usually bear significant influence on seed setting percentage. The height difference also played a major role in seed setting percentage. The height difference between the male and the female plants was the maximum of 54 cm in TNAU 18 S x IET 27044. The medium height difference was observed with the cross involved in the crosses of TNAU 18 S as female and the minimum height difference of 22.0 cm was observed in the hybrid generated form TNAU 60 S and CB-09–106. The height differences between the parents of ten hybrids during the flowering period showed notable influence in out-crossing percentage. The medium differences between two parents (22.8 cm to 51.20 cm) had great influences in the seed productions. This is in accordance with the statement elaborated by Virmani *et al,* [73] as this may be attributed to the appropriate height differences which might ensure the contacts of female line's stigma with the maximum pollens at peak anthesis period of the parents concerned.

The seeding interval was determined by the growth duration between the two parental lines. The one with longer duration was sown earlier according to the number of days of difference between the two parents in terms of days to 50 percent flowering [73]. Synchronizing period, though it did not have much variation in this study, had a little effect on the out-crossing potentials of these TGMS by exhibiting varying seed yield in different cross combinations. Three of four cross combination involving TNAU 18 S had the highest out-crossing. Cross combinations with TS-29–150 GY revealed the second highest out-crossing rate.

Manonmani [74] studied Tgms gene introgressed 200 lines forpollen fertility in plains (lowaltitude300 MSL) at Coimbatore during the summer. During the critical period of the crop growth the average temperature was 25–29°C. Then the selected lines were stubbleplanted in high altitude (1500MSL) at Hybrid Rice Evaluation Centre, Gudalur during Khariff. Average temperature of less than 20°C was recorded. During flowering stage, on microscopic observation with potassium iodide stain, some of the sterile lines recorded pollen free anthers (GDR 33S, TNAU 84S & TNAU 86S) and also differences in size of the pollens (GDR 29S). Pollen sterility level observed was 0–98%.All the seventy sterile stubbles from coimbatore were planted at Gudalur, fertility reversion rate was studied and selfed seeds were collected. Based on the pollen fertility observation the70 lines were grouped into four categories. Thirty eight lines showed >90% reversion, 13 lines showed 50–90% and < 11 lines showed <50% reversion and 8 lines showed no reversion. The Selfed seeds from the revesed lines were collected. These lines will be further exploited for their stability and will be used for the development of the two line hybrid in Tamil Nadu.

TGMS lines were also characterized with molecular markers. [75] investigated to study the genetic relationship of thermosensitive genic male sterile lines developed at Tamil Nadu Agricultural University, Coimbatore using morphological traits and SSR markers. Wide genetic variation among TGMS lines were observed for morphological and floral traits. SSR markers survey using 100 SSR markers revealed that 27 were polymorphic, amplifying a total of 71 alleles with an average of 2.67 alleles. TNAU 18S exhibited better performance based on the morphological characters, for a number of tillers per plant, angle of glume opening and panicle length and TNAU 45S expressed good floral characters. Cluster analysis differentiated six TGMS lines into four clusters.

Two TGMS lines (TNAU 60S and TNAU 95S) showed 100 per cent pollen and spikelet sterility and the remaining lines are in the range of 97–98 per cent pollen and spikelet sterility. The stable pollen sterility showed by TNAU 95S was also reported by Srimathi et al. [76] and Kanimozhi et al. [77]. There is a narrow variation in the angle of glume opening which ranged from 20 to 23° among the lines. Panicle length was observed to be more in TNAU 18S (20.25 cm) and while less in TNAU 39S (11 cm). There is a narrow variation in 100 seed weight ranged from 1.97 to 2.44 g. TNAU 45S had the highest stigma length (0.27 mm), stigma breadth (0.08 mm), anther length (0.31 mm) and anther breadth (0.06 mm) among all TGMS lines and TNAU 95S (0.13) had the highest pollen volume compared to all TGMS lines. Euclidean distance values ranged from 4.464 to 6.558 indicating the presence of a wide range of genetic diversity among the six TGMS lines. The ED value was maximum (6.558) between the genotypes TNAU 39S and TNAU 95S, indicating that these genotypes are diversely related to each other. Meanwhile, the minimum ED value was observed between genotypes TNAU 95S and TNAU 60S (4.464) and followed by genotypes TNAU 14S and TNAU 39S (4.507) indicating that these genotypes were closely related to each other.

Identification of the new polyploid rice photoperiod - and thermo-sensitive genic male sterile lines will provide material for further research into polyploidy and hybrid vigor in rice and promote the exploitation of polyploid hybrid rice [78].

Pardeep et al., [79] investigated the eighteen TGMS lines and being used for molecular characterization by sixteen SSR markers and correlated with critical sterility temperature. Based on the data generated on 18 TGMS lines, the UPGMA dendrogram was constructed using Jaccard's similarity coefficients. A total of 47 alleles were amplified using 16 SSR primer pairs. All the lines except for marker RM499 were found to be polymorphic. The range of alleles was 2–5, while the average number of alleles per primer was 2.93. All the three clusters contained one or two fertile lines in each namely, cluster I (TGMS-6), cluster II (TGMS-9) and (TGMS-18) and cluster III (TGMS-1). These fertile lines separated to other sterile lines by three markers with unique bands. TGMS-6 and TGMS-9 showed 200 bp

specific band by RM 324 marker, TGMS-1 and TGMS-18 showed 180 bp and 200 bp specific bands and in TGMS-1 also showed 180 bp specific band with RM 254 marker, it means that the specific bands 180 bp and 200 bp generated by different markers in different lines responsible for fertility.

Grouping of TGMA lines based on molecular markers were studied by Mengchen *et al.,* [80]. They studied 48 simple sequence repeat (SSR) markers, and genotyped a panel of 208 *indica* P/TGMS lines and confirmed three subgroups, named *indica*-I, *indica*-II and *indica*-III, in *indica* P/TGMS lines. Further diversity analysis indicated *indica*-II had the highest genetic diversity. The genetic differentiation between *indica*-II and *indica*-III was demonstrated as the largest among the three subgroups. Moreover, *indica*/*japonica* component identification was detected that five P/TGMS lines possess *indica* components less than 0.900. These results improve our knowledge on the genetic background for P/TGMS lines in China and will be beneficial for hybrid rice breeding programs.

TGMS Lines also introgressed with broad spectrum resistance for many diseases. Wang et al., [81] successfully bred the broad-spectrum resistance gene Xa23 through marker-assisted selection (MAS) combined with phenotypic selection in two novel inbred rice varieties and two photoperiod - and thermosensitive genic male sterility (P/TGMS) lines. All of the developed lines and derived hybrids exhibited enhanced resistance to BB with excellent yield performance.
