Philippine Journal of Crop Science April 2005, 30(1): 19-28 Copyright 2005, Crop Science Society of the Philippines Released 22 April 2005 IDENTIFICATION OF THERMOSENSITIVE GENIC MALE-STERILE LINES WITH LOW CRITICAL STERILITY POINT FOR HYBRID RICE BREEDING DL SANCHEZ & SS VIRMANI Plant Breeding, Genetics and Biochemistry Division (PBGB), International Rice Research Institute (IRRI), DAPO Box 7777, Metro Manila, Philippines The purity of hybrid rice seeds utilizing the 2-line system is increased when the thermosensitive genic male-sterile (TGMS) line possesses a low critical sterility point (CSP), which minimizes self-fertility induced by temperature fluctuations during hybrid rice seed production. To identify such TGMS lines at IRRI, Norin PL12, ID24, IR32364 (TGMS) and six IRRI-bred TGMS lines derived from Norin PL12 were sown in 3-week intervals from June to December 1999. Heading date, pollen fertility and spikelet fertility were obtained from each line in every planting interval. Correlation analyses between spikelet fertility and maximum and daily mean temperatures at 1-30 days before heading were done to determine the critical stage where temperature influences the sterility/fertility expression of the TGMS lines. The CSP values of the TGMS lines were identified through regression analyses between spikelet fertility and maximum and daily mean temperatures at the critical stage. The data showed that the critical stage for most TGMS lines occurs at the developmental stages between the differentiation of secondary branch primordium and the filling stage of pollen, or approximately 24 to 5 days before heading. Norin PL12, ID24, IR32364 (TGMS), IR72093 and IR72096 were identified to have low CSP (T max <32 C; T mean <27 C). The data indicates that evaluating for sterility in the wet season is more effective than in the dry season in identifying TGMS lines with low CSP. TGMS lines developed using this new strategy were found to be more stable in terms of sterility expression. Preliminary evaluation of 2-line hybrids from these low-csp TGMS lines showed a high frequency of heterotic combinations (77%). 2-line hybrid rice, critical stage, critical sterility point, hybrid rice breeding, sterility/fertility expression, TGMS INTRODUCTION Rice is a self-pollinated crop, a fact which renders hybrid breeding difficult and necessitates the management of fertility. The discovery of male sterility systems in rice has essentially resolved the constraint of controlling fertility/sterility and paved the way for the development of hybrids that have a 15-20% yield advantage over inbred high-yielding varieties. China pioneered the research in hybrid rice in 1964. In 1972, Chinese Professor Long Ping Yuan successfully developed the genetic material necessary for breeding hybrid rice and encouraged the growing of hybrid rice. From there, in 5 years, from 1976 to 2001, the accumulated area planted to hybrid rice in China increased to over 270 M ha, and the total increase in rice was 380 M tons (Ma & Yuan 2003). Other countries outside China have since also adapted hybrid rice technology. In 2002, the estimated area devoted to hybrid rice alone was 275,000 ha in India, 450,000 ha in Vietnam, 30,000 ha in the Philippines, 10,000 ha in Bangladesh and 5,000 ha in Indonesia. Some other countries have also been actively participating in hybrid rice research, and will be releasing hybrid rice commercially in the next few years (Virmani 2003). Currently, the cytoplasmic male sterility (CMS) system is the most widely used male sterility system for hybrid rice breeding. While effective and reliable, this system is cumbersome for hybrid seed production since CMS requires a third or maintainer line for seed multiplication of the male-sterile parent. In contrast, the environment-sensitive genic male sterility (EGMS) system requires only 2 lines. The EGMS makes use of photoperiod and/or temperature to control the sterility/fertility behavior of the female parent. Under certain conditions (usually short photoperiod and/or low temperature), the EGMS line is male-fertile; therefore, it
can be multiplied by selfing without the use of a maintainer line. Thus, aside from the simplification of EGMS multiplication, the 2-line system increases the frequency of heterotic hybrids, since any fertile line can be used as a male parent. The negative effects, if any, associated with sterility-inducing cytoplasm are also eliminated. EGMS systems are classified according to the environmental factors affecting the sterility/fertility expression: photoperiod-sensitive genic male sterility (PGMS), thermosensitive male sterility (TGMS), and photothermosensitive male sterility (PTGMS). The EGMS in rice first became possible when Nongken 58S was discovered in China by Shi (1985). This spontaneous mutant of the japonica variety Nongken 58 was male-sterile under longday conditions and reverted to fertility under short-day conditions. Since then, a number of PGMS and TGMS lines have been developed in China and outside China. In the tropics, where photoperiod differences are minimal but wide temperature differences exist among different altitudes or different seasons, it is more practical to use the TGMS system (Virmani & Ilyas-Ahmed 2001). One of the constraints in adapting 2-line hybrid rice breeding in commercial scale is the contamination of selfed seeds within the TGMS line resulting from temperature fluctuations occur during seed production. A sudden decrease in temperature at a sensitive period in panicle development would cause the TGMS line to revert to fertility, thus decreasing the purity of hybrid seeds. One of the strategies being employed to overcome this problem is to breed for TGMS lines with low critical sterility point (CSP). When used in hybrid seed production, TGMS lines possessing low CSP remain completely sterile despite sudden temperature changes during the sensitive growth stage. This eliminates selfing. The CSP is the critical temperature during the sensitive stage of a TGMS line that results in complete sterility. Hybrid rice breeders use either of the 2 parameters in determining the CSP of a TGMS line: maximum temperature or mean temperature, depending on geographical location. Viraktamath & Virmani (2001) have shown that it is the maximum temperature that influences the fertility/sterility expression of the TGMS line in the tropics. The ideal CSP for a TGMS line under tropical conditions is at maximum temperatures between 30 and 32 C (Ali et al 1995). In China, which is a temperate country, the daily mean temperature is used to identify the CSP of TGMS lines, since it is a function of both minimum and maximum temperatures (Mou et al 1998). The mean temperature is computed from 4 temperature readings at 20 6-hour intervals. The ideal CSP for a TGMS line is a daily mean temperature of 23-25 C (Ying 1999). The current study aims to screen TGMS lines with low CSP and establish the timing for a more efficient evaluation of TGMS lines in the tropics. MATERIALS & METHODS Nine TGMS lines were planted in single rows, with 12 plants per row, in concrete beds at IRRI. They were sown in 3-week intervals from 16 June to 1 December 1999. The lines used were 3 TGMS donors (ie, Norin PL12, IR32364S, ID24), and 6 lines derived from Norin PL12 (ie, IR68298S, IR68935S, IR68945S, IR71018S, IR72093S, and IR72096S). These lines are completely sterile when the maximum/minimum temperatures average 32.6 C/24 C during panicle development (April-May at IRRI). Dates of first and 50% heading were recorded. At the heading stage, 5-6 apical spikelets from the 2 main panicles per plant in each line were collected and fixed in 70% ethanol. The anthers were crushed and stained with 1% iodine-potassium iodide (I-KI) solution. Pollen fertility was observed under the microscope and was scored using the scale reported by Virmani et al (1997). The two main panicles were also covered in glassine bags at heading stage to insure selfing; this was intended for the measurement of the spikelet fertility due to selfing. The bagged panicles were harvested at maturity. The average of the spikelet fertility from the 2 bagged panicles per plant and the average spikelet fertility from the 12 plants were computed. Temperature data were obtained from the IRRI Climate Unit. Maximum temperatures and daily mean temperatures from 1-30 days before heading were obtained. This period corresponds to the panicle development of a rice plant from the differentiation of the first bract primordium to the time the spikelets are completely formed (Virmani & Sharma 1993). The daily mean temperature was computed from the average of the temperatures taken at 0200H, 0800H, 1400H and 2000H (Mou, personal communication). The sensitive stage of the TGMS lines was determined using the procedure described in Mou et al (1998). Correlations between spikelet fertility and maximum and daily mean temperatures from 1-30 days before heading were computed. The days having significant and negative correlations at 5% and 1% levels were noted. This period denotes the sensitive stage of the TGMS line. To establish the CSP of the TGMS lines, linear regression analyses between spikelet fertility and the average of the maximum and daily mean temperatures Low Critical Sterility Point For Hybrid Rice
during the sensitive stage were done. The CSP of a TGMS line is identified as the temperature corresponding to 0.5% spikelet fertility. This is the acceptable spikelet fertility rate for TGMS lines in their sterile phase (Lu et al 1998, Virmani et al 2002). The correlation and regression analyses were all done with Microsoft Excel. RESULTS & DISCUSSION Fertility behavior of TGMS lines The average spikelet fertility values of the 9 TGMS lines and the temperature data from August 1999 to March 2000 are shown in Table 1 and Table 2. Six TGMS lines showed complete spikelet fertility in at least two planting IR71018S, and IR72093S occurred when they flowered in mid-february or panicle initiation occurred from mid- January to early February (average T max = 27.9 C; average T ave = 24.7 C). Identification of critical stage & critical sterility point The critical stages where temperature influences the fertility/sterility expression of the TGMS lines were identified through correlation analyses between spikelet fertility and maximum and daily mean temperatures from 1 to 30 days before heading. The longest period in between abrupt changes in r values, and where most significant r values lay, was considered to be the critical Table 1. Spikelet fertility of nine TGMS lines, IRRI, 1999 wet season-2000 dry season TGMS Line Average Spikelet Fertility When Flowered in Aug Sep Oct Nov Dec Jan Feb Mar 16-31 1-15 16-30 1-15 16-31 1-15 16-30 1-15 16-31 1-15 16-31 1-15 16-29 1-15 Norin PL 12 0.2 - - 0.0-0.0 0.7 2.7-50.2 3.3 16.7 - - IR32364S - - - 0.0 0.0-0.0 0.5-0.5 0.8 2.0 0.0 - ID 24 - - 0.0 0.0 0.0-0.0-0.0 14.1 3.2-2.3 - IR68298-0.0 2.7 15.2-0.0-20.4-29.8 11.3 22.9 - - IR68935-14.8 0.9 15.4-3.0 9.0 12.2-24.2 19.3-22.0 - IR68945 - - - - 23.0-19.6 20.4 28.1 42.6-30.6-33.1 IR71018-7.0-6.5-7.9 18.7 17.0-32.0 9.5-38.6 - IR72093 - - 0.2 2.6 3.0 0.0-0.0 5.4-0.5 10.2 0.2 - IR72096 - - 3.0 0.0 0.0 0.0 3.9-2.6 5.7 1.4-0.2 - intervals between June 16 and December 1 1999. These lines were: Norin PL12, ID24, IR32364S, IR68298S, IR72093S and IR72096S. Most of the TGMS lines were sterile when they flowered in October and November, or when the panicle development occurred between September and October (average T max = 30.6 C; average T ave = 26.5 C). IR68935S, IR68945S, and IR71018S did not show complete sterility in all planting intervals. Most of the TGMS lines showed the highest spikelet fertility values when they flowered in January or when panicle development occurred in December (average T max = 28.2 C; average T ave = 25.0 C). Maximum spikelet fertility in IR32364S, stage of the TGMS line (Figure 1). The CSPs of the TGMS lines were identified through linear regression analyses between spikelet fertility and the average of the maximum and daily mean temperatures during the critical stage. For this study, only the maximum and daily mean temperatures were considered as parameters in determining the CSP of the TGMS lines. The minimum temperature was not used because Viraktamath & Virmani (2001) had shown that between the maximum and the minimum temperature, it is the maximum that influences the fertility/sterility expression of the TGMS lines, as already stated. DL Sanchez & SS Virmani 21
Figure 1a-i. Correlation coefficients between temperature and spikelet fertility of nine TGMS lines at 1-30 days before heading (continued next page) 22 Low Critical Sterility Point For Hybrid Rice
Figure 1a-i. Correlation coefficients between temperature and spikelet fertility of nine TGMS lines at 1-30 days before heading (continued next page) DL Sanchez & SS Virmani 23
Figure 1a-i. Correlation coefficients between temperature and spikelet fertility of nine TGMS lines at 1-30 days before heading (continued from previous page) 24 Low Critical Sterility Point For Hybrid Rice
Chinese scientists, on the other hand, use the daily mean temperature to identify the CSP of TGMS lines, since it is a function of both minimum and maximum temperatures (Mou et al 1998). The nine TGMS lines had varying sensitive stages where temperature affects their fertility/sterility behavior. Generally the sensitive stage occurs between the differentiation of secondary branch primordium and filling stage of pollen, or approximately 24 to 5 days before heading. They also have varying CSP, even if 7 of them (Norin PL12 and its derivatives, ie, IR68298S, IR68935S, IR68945S, IR71018S, IR72093S, and IR72096S), have the same male-sterile gene, tms2. The change in CSP when a TGMS gene is transferred to different backgrounds has also been observed by Mou et al (1998) and Virmani The critical stage where temperature influences fertility/sterility expression in IR32364S cannot be defined with the data available due to the low range of its spikelet fertility (0-2%). It is safe to say that this line has a low CSP; however, we need to identify a temperature at which this line can be multiplied before it can be used in 2-line hybrid rice production. Reassessment of TGMS evaluation procedures at IRRI Based on the results of this study, TGMS lines evaluated for sterility when maximum temperature is above 31 C may result in selecting those with high CSP. TGMS lines were previously evaluated at IRRI in this temperature regime, which occurs during the dry season in Table 2. Temperature at IRRI wetland, Aug 1999 to Feb 2000 T max ( o C) 27.7-32.6 T ave ( o C) 25.2-28.2 T min ( o C) 22.7-26.2 Aug Sep Oct Nov Dec Jan Feb 1-15 16-31 1-15 16-30 1-15 16-31 1-15 16-30 1-15 16-31 1-15 16-31 1-15 16-29 26.6-32.1 24.6-28.7 22.2-24.8 26.7-31.7 23.9-27.8 22.6-24.7 28.6-33.2 29.4-3325.6-32 25-31.6 26-30.925-31.2 24.4-30.5 25.2-28 25.3-28 24.1-27.5 22.7-24.5 22.4-25.1 23-25 20.7-24.8 23.8-27 24.1-26.6 22.6-24.6 23.9-26.6 22.8-26.5 23-25.1 21.5-24.5 28.1-30.9 26.8-25.1 21.2-24.5 25.3-30.6 22.4-26.3 19.2-22.6 24.5-3225-30.7 23.4-27.2 21.2-24.5 24.1-27.1 21.2-24 & Viraktamath (2001). The critical stages and CSP of the TGMS lines are summarized in Table 3. TGMS lines (ie, IR68945S, IR68935S and IR71018S) that did not show complete sterility at any point of the study were found to have CSPs above 32 C maximum temperature and 27 C daily mean temperature. The high CSP and sensitivity to temperature fluctuations of IR68945S was earlier reported by Viraktamath & Virmani (2001). In the phytotron, IR68945S reverted to partial fertility when interruptions of 27 C occurred for 2 hours at 32 C/24 C and 27 C for 4 hours even for 1 day in the critical stage. IR68298S, showing complete sterility in 2 nonconsecutive planting intervals, also had a relatively high CSP (31.5 C maximum temperature and 27.1 C daily mean temperature). Thus, this line, along with IR68945S, IR68935S, and IR71018S, is not recommended for use in hybrid rice seed production. April and May (Lopez & Virmani 2000). These lines reverted to fertility status when the temperature fluctuated below 30 C during the critical stage of panicle development. Evaluating the TGMS lines during the wet season, when panicle initiation to flowering occurs between August and September, is therefore considered a more effective way of identifying those with low CSP. Figure 2 shows the average daily maximum, minimum and mean temperatures at the IRRI wetland area in 1979-2000. Maximum temperatures during April and May were higher than those during August and September. Though maximum temperatures at IRRI between August and September are usually between 30-32 C, fluctuations below 30 C occur occasionally during cloudy and/or rainy weather. Thus, evaluating the TGMS lines for sterility under DL Sanchez & SS Virmani 25
these conditions would eliminate those with high CSP, while those that are found to be sterile are safer to use in hybrid rice seed production. Based on these findings, suspected TGMS lines obtained from sterile F 5 and F 6 in the TGMS pedigree nursery that reverted to fertility at 26/21 C in the phytotron were evaluated for sterility by sowing them in the first week of July. This practice started in 2000. With the change in sowing schedule of the evaluation nursery, TGMS lines with high CSP were identified and discarded. in the test cross nursery. The panicle weight data of hybrids vis-à-vis the better parent/check variety indicated 77% of the hybrids were heterotic, confirming the previous finding. CONCLUSIONS To develop tropical 2-line hybrids using the TGMS system, TGMS lines should have a CSP that is low enough to minimize self seed-setting in case of sudden Table 3. Critical stages and critical sterility points of nine TGMS lines TGMS Critical Stage CSP ( o C) Line Days Approximate developmental stage After PI Before heading T max T ave Norin PL 12 14-21 9-16 Formation stage of pollen mother cell to meiotic division IR32364S undefined ID 24 16-20 10-14 Formation stage of pollen mother cell to meiotic division IR68945 9-15 15-21 Differentiation of secondary branch primordia to formation stage of pollen mother cell IR68935 16-25 5-14 Formation stage of pollen mother cell to ripe stage of pollen IR68298 13-19 11-17 Formation stage of pollen mother cell to meiotic division IR71018 6-18 12-24 Differentiation of secondary branch primordia to meiotic division IR72093 14-22 8-16 Formation stage of pollen mother cell to meiotic division IR72096 15-19 11-15 Formation stage of pollen mother cell to meiotic division 30.6 26.5 29.5 25.9 34.1 28.8 32.4 27.7 31.5 27.1 32.2 27.4 30.4 26.3 31.3 26.9 Two promising TGMS lines (ie, IR73827-23S and IR73834S) were identified to have low CSP. They were sterile even when maximum temperature decreased to 28 C. The characteristics of these TGMS lines are shown in Table 4, while their grain quality traits are shown in Table 5. These lines are currently being used to make test crosses for preliminary evaluation of 2-line hybrids. Lopez & Virmani (2000) compared the frequency of heterotic hybrids in the 2-line and 3-line test cross nurseries at IRRI in 1993-1994. The frequency of heterotic hybrids based on the 2-line system was higher (36-67%) than that based on the 3-line system (16-31%). In the 2001 wet season, we also evaluated 35 additional 2-line hybrids 26 temperature decrease (maximum temperature of about 27-28 C). Fertility-inducing temperature (maximum temperature of at least 25 C) should also be consistently available at certain periods or in areas of higher elevation. The TGMS line should also have characteristics that commercially useful CMS lines should have, ie, wide adaptability, high outcrossing ability, good combining ability, and acceptable grain quality (Virmani et al 1997). Screening the TGMS lines for low CSP at IRRI is more effective in the wet season than in the dry season at IRRI. The TGMS lines IR73827-23S and IR73834S were identified using this modified timing in screening and were found suitable for use in developing 2-line hybrids. Low Critical Sterility Point For Hybrid Rice
LITERATURE CITED Ali J, Siddiq EA, Zaman FU, Abraham MJ & Ilyas Ahmed M. 1995. Identification and characterization of temperature sensitive male-sterile sources in rice (Oryza sativa L.). Indian Journal of Genetics 55(3): 243-259 Lopez MT & Virmani SS. 2000. Development of TGMS lines for developing 2-line rice hybrids for the tropics. Euphytica 114: 211-215 Ma GH & Yuan LP. 2003. Hybrid rice achievements and development in China. In Hybrid Rice For Food Security, Poverty Alleviation And Environmental Protection. Proceedings of the 4 th International Symposium on Hybrid Rice, Hanoi, Vietnam, 14-17 May 2002, Virmani SS, Mao CX & Hardy B (ed), International Rice Research Institute. Los Baños, Philippines. pp 247-256 Mou TM, Li CH, Young YC & Lu XG. 1998. Breeding and characterizing indica PGMS and TGMS lines in China. In Advances In Hybrid Rice Technology. Proceedings of the 3rd International Symposium on Hybrid Rice, 14-16 November 1996, Hyderabad, India, Virmani SS, Siddiq EA & Muralidharan K (ed), International Rice Research Institute. Los Baños, Philippines. pp 79-88 Shi MS. 1985. The discovery and study of the photosensitive recessive male-sterile rice (Oryza sativa L. subsp. Japonica). Scientia Agricultura Sinica 2: 44-48 Critical stage 34 33 32 Tmax Tmin Tave Temperature ( o C) 31 30 29 28 27 26 25 24 23 22 21 20 01- Oct 15- Oct 29- Oct 12- Nov 26- Nov 10- Dec 24- Dec 07- Jan 21- Jan 04- Feb 18- Feb 03- Mar 17- Mar 31- Mar Date 14- Apr 28- Apr 12- May 26- May 09- Jun 23- Jun 07- Jul 21- Jul 04- Aug 18- Aug 01- Sep 15- Sep 29- Sep Figure 2. Average temperature at IRRI wetland, 1979-2000 Viraktamath BC & Virmani SS. 2001. Expression of thermosensitive genic male sterility in rice under varying temperature situations. Euphytica 122: 137-143 Virmani SS. 2003. Advances in hybrid rice research and development in the tropics. In Hybrid Rice For Food Security, Poverty Alleviation And Environmental Protection. Proceedings of the 4 th International Symposium on Hybrid Rice, Hanoi, Vietnam, 14-17 May 2002, Virmani SS, Mao CX & Hardy B (ed), International Rice Research Institute. Los Baños, Philippines. pp 7-20 Virmani SS & Ilyas-Ahmed M. 2001. Environment-sensitive genic male sterility (EGMS) in crops. Advances In Agronomy 72: 139-195 Virmani SS & Sharma HL. 1993. Manual For Hybrid Rice Seed Production. International Rice Research Institute, Los Baños, Philippines. 57 pp DL Sanchez & SS Virmani 27
Table 4. Characteristics of promising low-csp TGMS lines TGMS Line Pedigree Days from Sowing to Heading Phenotypic Acceptability a Seed Setting b Out-crossing Potential (%) IR73827-23S ID 24/ IR 64 90 3 5-7 24.39 IR73834S ID 24/ IR58025B 87 5 3-5 40.91 a In a scale of 1-9; 1=excellent and 9=unacceptable. b In a scale of 1-9; 1=; 1=highly fertile (>90% spikelet fertility) and 9=completely sterile (0% spikelet fertility). Table 5. Grain quality traits of promising low-csp TGMS lines Grain Quality Traits TGMS Line Pedigree Length c Shape d Chalkiness e Gelatinization Temperature Amylose Content (%) IR73827-23S ID 24/ IR 64 5 5 5 HI/I 20.4 IR73834S ID 24/ IR58025B 3 1 0 L 16.2 c In a scale of 1-7; 1= Extra long (more than 7.5 mm); 7= Short (5.5mm or less). d In a scale of 1-9; 1=slender, 9=round. e In a scale of 0 to 9; 0=None, 9=Large (more than 20%) Virmani SS, Sun ZX, Mou TM, Jauhar Ali A & Mao CX. 2003. 2-Line Hybrid Rice Breeding Manual. International Rice Research Institute, Los Baños, Philippines. 88 pp Virmani SS, Viraktamath BC, Casal CL, Toledo RS, Lopez MT & Manalo JO. 1997. Hybrid Rice Breeding Manual. International Rice Research Institute, Los Baños, Laguna, Philippines. 151 pp Ying HQ. 1999. Scientific progress made in 2-line system hybrid rice research and production in China. Hunan Agricultural Research Newsletter 6(3): 4-8 28 Low Critical Sterility Point For Hybrid Rice