Thermo-sensitive Genic Male Sterile Rice Research Articles

Rapid generation of fragrant thermo-sensitive genic male sterile rice with enhanced disease resistance via CRISPR/Cas9.

The three, by mutagenesis produced genes OsPi21, OsXa5, and OsBADH2, generated novel lines exhibiting desired fragrance and improved resistance. Elite sterile lines are the basis for hybrid rice breeding, and rice quality and disease resistance become the focus of new sterile lines breeding. Since there are few sterile lines with fragrance and high resistance to blast and bacterial blight at the same time in hybrid rice production, we here integrated the simultaneous mutagenesis of three genes, OsPi21, OsXa5, and OsBADH2, into Zhi 5012S, an elite thermo-sensitive genic male sterile (TGMS) variety, using the CRISPR/Cas9 system, thus eventually generated novel sterile lines would exhibit desired popcorn-like fragrance and improved resistance to blast and bacterial blight but without a loss in major agricultural traits such as yield. Collectively, this study develops valuable germplasm resources for the development of two-line hybrid rice with disease resistance, which provides a way to rapid generation of novel TGMS lines with elite traits.

Estimation of the Thermosensitive Period of the Thermosensitive Genic Male Sterile Rice Line ‘Norin-PL12’

Estimation of the Thermosensitive Period of the Thermosensitive Genic Male Sterile Rice Line ‘Norin-PL12’

Reproductive tissue-specific translatome of a rice thermo-sensitive genic male sterile line

Translational regulation, especially tissue- or cell type-specific gene regulation, plays essential roles in plant growth and development. Thermo-sensitive genic male sterile (TGMS) lines have been widely used for hybrid breeding in rice (Oryza sativa). However, little is known about translational regulation during reproductive stage in TGMS rice. Here, we use translating ribosome affinity purification (TRAP) combined with RNA sequencing to investigate the reproductive tissue-specific translatome of TGMS rice expressing FLAG-tagged ribosomal protein L18 (RPL18) from the germline-specific promoter MEIOSIS ARRESTED AT LEPTOTENE1 (MEL1). Differentially expressed genes at the transcriptional and translational levels are enriched in pollen and anther-related formation and development processes. These contain a number of genes reported to be involved in tapetum programmed cell death (PCD) and lipid metabolism during pollen development and anther dehiscence in rice, including several encoding transcription factors and key enzymes, as well as several long non-coding RNAs (lncRNAs) that potentially affect tapetum and pollen-related genes in male sterility. This study represents the comprehensive reproductive tissue-specific characterization of the translatome in TGMS rice. These results contribute to our understanding of the molecular basis of sterility in TGMS rice and will facilitate further genetic manipulation of TGMS rice in two-line breeding systems.

Differential expression of transport and signalling genes in leaves and panicle regulates the development of pollen-free anthers in TGMS red rice

Thermosensitive genic male sterile (TGMS) plants are male sterile above a critical sterility temperature (CST) and become male fertile below CST during a critical thermosensitive stage. It is essential to analyse the gene expression pattern under fertility- and sterility-inducing conditions to understand the mechanisms of male sterility since it is regulated by a single recessive nuclear gene sensitive to environment during a specific stage of panicle development. Hence, this study aims at understanding the molecular mechanism associated with pollen sterility in TGMS system. The newly developed TGMS line on red rice background exhibited male sterility with pollen-free anthers and hence can be recommended as a stable female parent for the development of suitable red rice hybrids to the state of Kerala. Microarray gene expression analysis of TGMS leaf and young panicle revealed that occurrence of pollen-free anthers in the TGMS line during sterility-inducing condition is mainly due to the down-regulation of genes encoding ABC transporter proteins, lipid transfer protein and strictosidine synthase, glucose-methanol-choline oxidoreductase, male sterility protein 2, wax synthase and β-1,3-glucanase. Moreover, pathways involved in the carbohydrate synthesis and transport were also down-regulated during sterility condition. The differential expression of transport and signalling genes regulates the development of pollen-free anthers in TGMS rice.

Comparative transcriptome analysis reveals the mechanism of exogenous substances inhibiting fertility alteration induced by low temperature in TGMS rice line

During two-line hybrid rice seed production, thermo-sensitive genic male sterile (TGMS) rice lines are susceptible to low temperature, leading to fertility alteration (from sterile to fertile) and production of false hybrids (self-pollination). To explore appropriate solutions to reduce the chance of seed production failure, two exogenous substances, d-Arg and ethephon, were applied to restrain the fertility alteration induced by low temperature (22.0 °C), respectively. The fertile pollen grains and bagged seed setting rate of TGMS rice line Jing 4155S (with the critical fertility/sterility alteration temperature of 23.5 °C) under low temperature could be reduced to 0% by spraying exogenous substances. Transcriptome analysis of young panicles of TGMS rice line Jing 4155S demonstrated that the expressions of multiple transcription factors (MYB, FAR1, and ERF, etc.) changed, and hence affecting various downstream related genes and metabolic processes, including hormonal signal transduction, plant pathogen interactions, circadian rhythm pathway, carbohydrate metabolism, cell wall synthesis, and lipid metabolism, etc. The enhanced ethylene biosynthesis and the prevented redox equilibrium from unbalance to balance in young panicles were one of the major reasons that fertility alteration was inhibited by exogenous substances. Our results made a contribution to the solution for inhibiting fertility alteration induced by low temperature in two-line TGMS rice lines during hybrid seed production.

Generation of a new thermo-sensitive genic male sterile rice line by targeted mutagenesis of TMS5 gene through CRISPR/Cas9 system

BackgroundTwo-line hybrid rice with high yield potential is increasingly popular and the photo- and temperature-sensitive male sterile line is one of the basic components for two-line hybrid rice breeding. The development of male sterile lines through conventional breeding is a lengthy and laborious process, whereas developing thermo-sensitive genic male sterile (TGMS) lines for two-line hybrid breeding by editing a temperature-sensitivity gene by CRISPR/Cas9 is efficient and convenient.ResultsHere, thermo-sensitive genic male sterility (TGMS) was induced by employing the CRISPR/Cas9 gene editing technology to modify the gene TMS5. Two TGMS mutants, tms5–1 and tms5–2, both lacking any residual T-DNA, were generated in the indica rice cultivar Zhongjiazao17 (cv. YK17) background. When grown at a sub-optimal temperature (22 °C), both mutants produced viable pollen and successfully produced grain through self-fertilization, but at temperatures 24 and 26 °C, their pollen was sterile and no grain was set. F1 hybrids derived from the crosses between YK17S (tms5–1) and three different restorer lines outperformed both parental lines with respect to grain yield and related traits.ConclusionThe YK17S generated by CRISPR/Cas9 system was proved to be a new TGMS line with superior yield potential and can be widely utilized in two-line hybrid breeding of indica rice.

The transcription factor OsbHLH138 regulates thermosensitive genic male sterility in rice via activation of TMS5.

Thermosensitive genic male sterile (TGMS) lines favored heterosis exploitation in two-line hybrid rice. TMS5, a member of RNase Z cleavages the UbL40 mRNAs, plays an important role in two-line hybrid rice. Here, we identified a new TGMS mutant 93-11s, which lost two amino acids in the first exon of TMS5 gene and caused thermosensitive genic male sterility in rice. The tms5-2 cannot process mRNAs of the ubiquitin fusion ribosomal protein L40 (UbL40) and hence cause the mRNAs accumulation in restrictive temperature. Further, we identified a nucleus-localized bHLH transcription factor OsbHLH138, which can form the basic helix-loop-helix structure and bind the core region of tms5-2 promoter sequences by bHLH domain, and activate expression of tms5-2 by the acidic amino acid-rich domain. These results indicate a novel mechanism for the tms5-2 regulating thermosensitive male sterility of rice. By altering expression of OsbHLH138, we can regulate the expression level of TMS5 and the accumulation of UbL40 mRNAs to command the male fertility in different temperatures. The identification of OsbHLH138 provides breeders a new choice for development of TGMS rice lines, which will favor the sustainable development of two-line hybrid rice.

Association between sequence variants in cadmium-related genes and the cadmium accumulation trait in thermo-sensitive genic male sterile rice

Although cultivation of hybrid rice varieties has been increasing, there are risks that high levels of cadmium (Cd) will accumulate in grain when such rice is grown in Cd-polluted environments. To produce Cd-safe hybrid rice, one practical approach is the generation of low Cd-accumulating parental lines. In two-line hybrid breeding, thermosensitive genic male sterile (TGMS) lines function as female parents to yield hybrid seeds. Recently, Cd accumulation-related genes have been identified; however, the effect of these genes on Cd accumulation in the grains of TGMS lines has yet to be reported. Here, 174 TGMS lines were selected for Cd accumulation phenotyping, and 30 TGMS lines, including 15 stable low-Cd and 15 high-Cd lines, were selected for single-nucleotide polymorphism (SNP) genotyping and association analysis. Association studies were conducted to identify the relationship between Cd accumulation and variable sites within seven candidate Cd-associated genes using logistic models. Nine sequence variant sites in four of the candidate genes were found to be significantly associated with Cd accumulation, two of which in OsNRAMP1 and OsNRAMP5 are low-Cd favorable variants, explaining 46.4% and 22.6% of the phenotypic variation, respectively. These loci could be developed as new molecular markers for identification of Cd accumulation characteristics and low-Cd marker-assisted breeding.

Progress on photoperiod thermo-sensitive genic male sterile rice

Hybrid rice has been cultivated worldwide since Yuan LongPing initially proposed the idea of developing and applying hybrid rice in rice production 50 years ago. Hybrid rice has provided a strong guarantee for the food security in China. The application of hybrid rice was firstly based on cytoplasmic male sterility using a 3-line system, including the sterile line, the maintainer line, and the restorer line. Later, the discovery of some environment-sensitive genic male sterile lines whose fertility changed in different environments, indicated their strong application potential in hybrid rice production. Shi MingSong accidentally found a natural male sterile mutant Nongken 58S in the field when planting normal japonica variety Nongken 58. The pollen of Nongken 58S was sterile under long-day conditions, while was fertile under short-day conditions. Based on this special property, Nongken 58S could be developed as a photoperiod-sensitive genic male sterile line for hybrid rice: it would be a sterile line at longdays, meanwhile served as a maintainer line at shortdays. Since then, the theory of 2-line system has been improving. Furthermore, the discovery of some other thermosensitive genic male sterile lines such as 5460S, Annong S-1 and Hengnong S-1 enriched this system. They were sterile under high temperatures, but converted to fertility under low temperatures. The 2-line hybrid rice based on the photoperiod thermo-sensitive genic male sterile lines has played important roles in the hybrid rice production. From 1996 to 2013, the proportion of 2-line hybrid rice increased from 0.92% to 33.59%. Some early genetic analysis indicated that the fertility of most photoperiod thermo-sensitive genic male sterile lines was controlled by a recessive allele. And the genetic linkage map of these loci has been established. But only until recent years, with the completion of rice genome and the development of new sequencing technologies and rice transgenic techniques, several genes controlling photoperiod- sensitive genic male sterility or thermo-sensitive genic male sterility in rice have been cloned. Among them, pms3 was the first reported, the mutation of which caused Nongken 58S. pms3 encodes a long non-coding RNA LDMAR. A SNP of LDMAR altered its RNA secondary structure, resulting in increased siRNA-directed DNA methylation in the promoter, which reduced the expression of LDMAR in Nongken 58S under long-day conditions. Another group found that the SNP was located in a siRNA (termed as osa-smR5864m); loss-of-function of this siRNA may fail to suppress the expression of its target genes. Shortly after, tms5 , a thermosensitive genic male sterility gene in Annong S-1 was charactered to encode RNase ZS1 processing Ub L40 mRNAs. Overaccumulation of Ub L40 mRNAs resuting from a premature stop codon of RNase ZS1 gene created by a SNP in the tms5 mutant caused the male sterility under high temperatures. Elucidation of the molecular mechanism of these genes have important implications for breeding new types of excellent and stable two-line male sterile lines in the future.

Molecular marker-assisted pyramiding of broad-spectrum disease resistance genes, Pi2 and Xa23, into GZ63-4S, an elite thermo-sensitive genic male-sterile line in rice

The two-line system of hybrid rice seed production based on thermo-sensitive genic male-sterile (TGMS) lines is much more cost-effective, simple and efficient than the three-line system with cytoplasmic male sterility. In this study, we report the introgression of a rice blast resistance gene Pi2 from VE6219 and a bacterial blight (BB) resistance gene Xa23 from HBQ810 into Guangzhan63-4S (GZ63-4S), an elite TGMS rice line, through marker-assisted pyramiding, leading to the development of Hua1015S, a blast- and BB-resistant pyramided line. The newly developed TGMS line Hua1015S and its derived hybrids showed resistance to rice blast and BB. Hua1015S had a similar critical temperature point of fertility–sterility alteration to GZ63-4S and similar key agronomic and grain quality traits. However, Hua1015S gave higher hybrid seed yields on account of increased outcrossing rates due to better floral traits, e.g. spikelet dehiscence, angle of glume opening, and stigma exsertion. The newly developed TGMS parental line Hua1015S with Pi2 and Xa23 is currently being utilized for heterosis breeding of broad-spectrum blast- and BB-resistant two-line rice hybrids and as an improved disease donor source for further TGMS parental line improvement.

Elongation of the uppermost internode for Shuangdi Pei eS, TGMS rice with eui gene

Shuangdi Pei eS, a thermo-sensitive genic male sterile (TGMS) rice line with eui gene is derived from TGMS rice line Shuangdi S by irradiation with 350 Gy60Co γ-ray. To elucidate the uppermost internode elongation of TGMS line with eui gene, Shuangdi Pei eS and its original TGMS line Shuangdi S were used to study the effects of temperature on panicle exsertion. At 25°C, the uppermost internode of Shuangdi Pei eS had the quickest elongation from the 4th day before flowering to 0 day (flowering), being 2.1-fold as that of Shuangdi S, whereas it elongated slowly during the 12th day to the 4th day before flowering and the 1st to the 3rd day after flowering. The uppermost internode of Shuangdi Pei eS exserted from the flag leaf sheath at 23, 25 and 27°C, respectively, and the length of elongated uppermost internode increased with the decreasing temperatures. At 30°C, though the panicles of Shuangdi Pei eS were still enclosed in the leaf sheath, the degree of panicle enclosure was significantly lower compared with Shuangdi S. Cytological studies on Shuangdi Pei eS which showed that the uppermost internode elongation was attributed to the increase of cell number and cell elongation, and the latter was more significant. Moreover, the numbers of outermost and innermost parenchyma cells and the cell length of the uppermost internode reduced with the increasing temperatures. Key words: Rice (Oryza sativa L.), thermo-sensitive genic male sterility (TGMS), elongated uppermost internode gene (eui), panicle exsertion, temperature.

Development and Application of a Computer-aided Selection System for Thermo-sensitive Genic Male Sterile Rice Multiplying Site

Great success has been achieved in the utilization of two-line hybrid rice in China.Nevertheless,the seed production of two-line hybrid rice is still a key technology that limited the extension of two-line hybrid rice on large scale.Seed multiplication of the thermo-sensitive genic male sterile (TGMS) rice,which with a narrow range of temperature for fertility restoration,is hard to be success by using the most common multiplying technologies currently.In order to improve the yield stability and security in TGMS rice multiplication,we developed a practical computer selection system for choosing an optimal multiplying site.This system using the java language was able to select suitable multiplying sites with optimal ecological factors which meet the requirement of the fertility sensitive stage (FSS) and heading-flowering stage (HFS) for TGMS rice multiplication.By using this computer-aided selection system,24,29,20,21,and 22 ecological areas were selected,which were superior to the most common seed producing area,Sanya of Hainan province,where the suitable probabilities of FSS and HFS are 35.0%,42.5%,55.0%,65.0%,and 75.0% for the TGMS with the critical temperature of 22.0℃,22.5℃,23.0℃,23.5℃,and 24.0℃,respectively,while the suitable probability of the optimal multiplying site selected by the computer-aided selection system were 83%,93%,100%,100%,and 100% in both FSS and HFS for TGMS with different critical temperature,respectively,showing much higher values than those in Sanya,Hainan Province.In 2010,we tried to produce the seed of C815S,a TGMS rice with the critical temperature of 22.0℃ in Baoshan,Yunnan Province selected by the computer-aided selection system.The grain yield reached 8 437.5 kg ha-1,and created the highest yield record of seed multiplication of the TGMS rice.These results suggested that the problems of unstable yield and low security for the TGMS rice seed multiplication should be solved by this computer aided selection system,and we believe the optimal multiplying site selected by the computer-aided selection system will provide a useful technical support for healthy and sustainable development in the two-line hybrid rice.

Thoughts and Practice on Some Problems about Research and Application of Two-Line Hybrid Rice

The main problems about research and application of two-line hybrid rice were reviewed, including the confusing nomenclature and male sterile lines classification, the unclear characteristics of photoperiod and temperature responses and the unsuitable site selection for male sterile line and hybrid rice seed production. In order to efficiently and accurately use dual-purpose genic male sterile lines, four types, including PTGMS (photo-thermo-sensitive genic male sterile rice), TGMS (thermo-sensitive genic male sterile rice), reverse PTGMS and reverse TGMS, were proposed. A new idea for explaining the mechanism of sterility in dual-purpose hybrid rice was proposed. The transition from sterile to fertile was involved in the cooperative regulation of major-effect sterile genes and photoperiod and/or temperature sensitive ones. The minor-effect genes with accumulative effect on sterility were important factors that affected the critical temperature of sterility transfer. In order to make better use of dual-purpose lines, the characterization of responses to photoperiod and temperature of PTGMS should be made and the identification method for the characterization of photoperiod and temperature responses of PTGMS should also be put forward. The optimal ecological site for seed production could be determined according to the historical climate data and the requirements for the meteorological conditions during the different periods of seed production.

Isolation and Characterization of a Microsporocyte-Specific Gene, OsMSP, in Rice

A spikelet-specific cDNA clone, OsMSP (Oryza sativa meiosis serine protease), was isolated from a rice cDNA library constructed by suppression subtraction hybridization (SSH). Its expression level was significantly reduced in the thermo-sensitive genic male-sterile rice line at sterile temperature (30/26°C, day/night) compared with that at fertile temperature (23/21°C, day/night) conditions. In contrast, no significant differences were observed in the normal fertile line under the above two sets of temperature conditions. The cDNA is 2,867 bp in length with an open reading frame (ORF) encoding 837 amino acid residues. Northern blots revealed that OsMSP transcripts were specifically present in spikelets at meiosis, and reached the highest level at early microspore stage. mRNA in situ hybridization confirmed that OsMSP was a rice microsporocyte-specific gene and its expression was limited to early microspore developmental stage. BLAST and phylogenetic analyses revealed that OsMSP shared high similarities with subtilisin-like serine protease genes from other organisms at both nucleotide and amino acid levels.

Comparative analysis of young panicle proteome in thermo-sensitive genic male-sterile rice Zhu-1S under sterile and fertile conditions

Proteome analysis was carried out to identify the young panicle proteins during different developmental stages under sterile and fertile conditions. Based on spot quantity and quality, 50 protein spots were analyzed by matrix associated laser desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS) and 20 spots were identified. Most of these proteins are closely associated with energy metabolism, protein biosynthesis, cell wall formation and stress responses, which are essential cell activities to the pollen development. Gene expression analysis of three different proteins by semi-quantitative RT-PCR showed that the mRNA level was not correlated exactly with the protein level.

Plant Temperature and Its Simulation Model of Thermo-Sensitive Genic Male Sterile Rice

Plant temperature ( T p) and its relations to the microclimate of rice colony and irrigation water were studied using a thermo-sensitive genic male sterile (TGMS) rice line, Pei'ai 64S. Significant differences in the daily change of temperature were detected between T pand air temperature at the height of 150 cm ( T A). From 8:00 to 20:00, T p was lower than T A, but they were similar during 21:00 to next 7:00. The maximum T p occurred one hour earlier than the maximum T A, though they both reached the minimum at 6:00. T p fluctuated less than T A. At the same height, during 6:00–13:00, T p was higher than air temperature ( T a), and T p reached the maximum one hour earlier than T a. During the rest time on sunny day, T pwas close to or even a little lower than T a. On overcast day, T pwas higher than T a in the whole day, and both maximized at the same time. In addition, T pwas regulated by solar radiation, cloudage and wind speed in daytime, and by irrigation water at night. The present study indicated that a T A of 29.6°C was the critical point, at which T pwas increased or decreased by irrigation water. T p and the difference between water and air temperatures showed a conic relation. T p fluctuation was also regulated by the absorption or reflection of solar radiation by leaves during daytime and release of heat energy during nighttime. By analysis on correlation and regression simulation, two models of T p were established.

Elongation of the Uppermost Internode for Changxuan 3S, a Thermo-Sensitive Genic Male Sterile Rice Line

Changxuan 3S, a thermo-sensitive genic male sterile (TGMS) rice line with eui gene, is derived from the TGMS rice line Pei'ai 64S by irradiation with 350 Gy of 60Co γ-ray. To elucidate the uppermost internode elongation of the TGMS line with eui gene, Changxuan 3S and its parent Pei'ai 64S were used to study the effects of temperature on panicle exsertion. At 24°C, the uppermost internode of Changxuan 3S elongated the fastest from the 4 th day before flowering to 0 day (flowering), being 2.1-fold as that of Pei'ai 64S, whereas it elongated slowly during the 12 th day to the 4 th day before flowering and the 1 st to the 3 rd day after flowering. The uppermost internode of Changxuan 3S exserted from the flag leaf sheath at 22°C, 24°C and 26°C, and the length of elongated uppermost internode increased with the decreasing temperatures. At 28°C, though the panicles of Changxuan 3S were still enclosed in the leaf sheath, the degree of panicle enclosure was significantly lower compared with Pei'ai 64S. Cytological studies on Changxuan 3S showed that the uppermost internode elongation was attributed to the increase of cell number and cell elongation, and the latter was more significant. Moreover, the numbers of outermost and innermost parenchyma cells and the cell length of the uppermost internode reduced with the increasing temperatures.

Improving Blast Resistance of a Thermo-Sensitive Genic Male Sterile Rice Line GD-8S by Molecular Marker-Assisted Selection

The broad-spectrum blast resistance gene Pi-1, from donor line BL122, was introduced into a thermo-sensitive genic male sterile rice line GD-8S, which possessed good grain quality but high susceptibility to rice blast, by using backcross breeding and molecular marker-assisted selection. Five elite improved male sterile lines, RGD8S-1, RGD8S-2, RGD8S-3, RGD8S-4 and RGD8S-5, were selected based on the results of molecular marker analysis, spikelet sterility, recovery rate of genetic background and agronomic traits. Thirty-three representative blast isolates collected from Guangdong Province, China were used to inoculate the improved lines and the original line GD-8S artificially. The resistance frequencies of the improved lines ranged from 76.47% to 100%, much higher than that of the original line GD-8S (9.09%). On the agronomic characters, there were no significant differences between the improved lines and GD-8S except for flag leaf length and panicle number per plant. The improved lines could be used for breeding hybrid rice with high blast resistance.

Characterization and identification of the candidate gene of rice thermo-sensitive genic male sterile gene tms5 by mapping

Previous research has demonstrated that the thermo-sensitive genic male-sterile (TGMS) gene in rice was regulated by temperature. TGMS rice is important to hybrid rice production because the application of the TGMS system in two-line breeding is cost-effective, simple, efficient and overcomes the limitations of the cytoplasmic male sterility (CMS) system. AnnongS is the first discovered and deeply studied TGMS rice line in China. Previous studies have suggested that AnnongS-1 and Y58S, two derivative TGMS lines of AnnongS, were both controlled by a single recessive gene named tms5, which was genetically mapped on chromosome 2. In the current study, three populations (AnnongS-1 x Nanjing11, Y58S x Q611, and Y58S x Guanghui122) were developed to investigate the tms5 gene molecular map. Analysis of recombination events of sterile samples, utilizing 125 probes covering the tms5 region, suggested that the tms5 gene was physically mapped to a 19 kb DNA fragment between two markers, 4039-1 and 4039-2, located on the BAC clone AP004039. Following the construction of a physical map between the two markers, ONAC023, a member of the NAC (NAM-ATAF-CUC-related) gene family, was identified as the candidate of the tms5 gene.

HPLC profiles of polyamines in the leaves and anthers of a new thermosensitive genic male sterile rice line

Hybrid rice is successfully cultivated in India and, in recent years, the country has released commercial rice hybrids having at least 1 t ha -1 higher yield than inbred checks. The cytoplasmic male sterility (CMS) system, also known as the three-line system, is presently the most widely used system for producing F 1 rice hybrids. Although effective, the system is cumbersome because CMS lines require specific maintainer and restorer lines and possess wild abortive (WA) cytoplasm that is vulnerable to biological stresses. On the other hand, the thermosensitive genic male sterility (TGMS) system does not require restorer lines, and the seeds of TGMS lines are multiplies by selfing when exposed to the right temperature at the critical growth stage (in the tropics, 5-15 d after panicle initiation) (Maruyama et al 1990).