Thermo-sensitive Genic Male Sterile Line Research Articles

Identification and Analysis of MS5d: A Gene That Affects Double-Strand Break (DSB) Repair during Meiosis I in Brassica napus Microsporocytes.

Here, we report the identification of the Brassica-specific gene MS5d, which is responsible for male sterility in Brassica napus. The MS5d gene is highly expressed in the microsporocyte and encodes a protein that localizes to the nucleus. Light microscopy analyses have demonstrated that the MS5d gene affects microsporocyte meiosis in the thermosensitive genic male sterility line TE5A. Sequence comparisons and genetic complementation revealed a C-to-T transition in MS5d, encoding a Leu-to-Phe (L281F) substitution and causing abnormal male meiosis in TE5A. These findings suggest arrested meiotic chromosome dynamics at pachytene. Furthermore, immunofluorescence analyses showed that double-strand break (DSB) formation and axial elements were normal but that DSB repair and spindle behavior were aberrant in TE5A meiocytes. Collectively, our results indicate that MS5d likely encodes a protein required for chromosomal DSB repair at early stages of meiosis in B. napus.

Critical stages of thermo-sensitive genic male sterile lines of rice in Kerala

Two line breeding is a viable proposition in a state like Kerala where rice is cultivated from below mean sea level to altitudes of 1500 MSL. The system provides a much simpler and economic hybrid seed production and broader choice of male parents for enhancing yield potential. A set of seven promising Temperature Sensitive Genic Male Sterile lines received from Tamil Nadu Agricultural University, Coimbatore, India viz., TGMS 74S, TGMS 81S, TGMS 82S, TGMS 91S, TGMS 92S, TGMS 93S and TGMS 94S were characterized for their sterility, fertility behavior under field conditions. Based on sterility/fertility behavior the entire study period of 2011 March to 2012 March can be grouped as two distinct periods viz, pollen sterile and pollen fertile months. The pollen sterile months indicated April and May when 100 per cent sterility was observed. July and August can be considered as pollen fertile months. Other months exhibited un stable expression of sterility/fertility and hence could not be grouped under pollen sterile or pollen fertile months. The sterile period was longest in TGMS 82S (87 days) followed by TGMS 81S (83 days). Since all these lines showed completely sterile for more than 30 consecutive days during sterile phase, these lines were successfully utilized in hybrid rice breeding programme. The Critical Sterility Temperature ranged from 34.20C (TGMS 74S, TGMS 91S, and TGMS 94S) to 38.6 0C (TGMS92S). Critical fertility temperature ranged from 23.6 0C (TGMS 91S) to 25.40C (TGMS 92S, TGMS 93S).

Development and validation of a functional co-dominant SNP marker for the photoperiod thermo-sensitive genic male sterility pms3 (p/tms12-1) gene in rice.

The fertility conversion of photoperiod thermo-sensitive genic male sterility (P/TGMS) lines in rice is mostly regulated by the P/TGMS genes in different environmental conditions. A point mutation with G-C on the pms3 (p/tms12-1) gene regulates the pollen fertility of Nongken58S and a large amount of Nongken58S-derived lines. In this study, we developed and designed a functional co-dominant marker according to the SNP loci for the pms3 (p/tms12-1) gene. We can differentiate the SNP loci in pms3 (p/tms12-1) gene from another TGMS lines and inbred cultivars using the dpms3-54 marker. The results showed that 376 bp band was detected in the homozygous genotype for pms3 (p/tms12-1), while 359 bp band was detected in the homozygous genotype for non-pms3 (p/tms12-1), two bands with 376 and 359 bp were detected in the heterozygous genotype. The dpms3-54 marker can be used to test the purity of two-line hybrid rice seeds and to divide each of F1 plant into homozygous and heterozygous genotypes at the seedling stage. Thus, this study provide a useful functional marker to detect pms3 (p/tms12-1) gene in different genetic resources and populations, which can be applied to the development and breeding of P/TGMS lines in two-line hybrid rice system with marker-assisted selection.

利用花药和未受精子房培养技术创建二倍体光温敏雄性核不育系与恢复系

[目的] 利用远缘杂交和多倍体双重优势选育水稻新品种是育种的新途径。在选育出多倍体减数分裂稳定性品系，解决多倍体结实率低的瓶颈问题后，开展多倍体水稻的理论和育种实践迫切需要合适的育种材料。[方法] 本文以多倍体水稻光温敏雄性核不育系PS001和多倍体水稻恢复系PR-T1为实验材料，分别对其进行花药和未受精子房培养而得到二倍体。[结果] 从而构建成二倍体和四倍体配套的光温敏水稻雄性核不育系和恢复系材料。[结论] 证明了利用花药和未受精子房培养既可以快速纯化品种，缩短性状稳定年限，达到快速育种的目的，又可以创造合适的用于理论研究的材料。 [Objective] Using double heterosis of polyploidization and distant hybridization to select new rice varieties is a new breeding strategy. After breeding the polyploid meiosis stability line and solving the bottleneck problem of low seed setting rate of polyploidy, the theory and breeding practice of polyploid rice urgently need suitable breeding materials. [Method] In this paper, the photoperiod and thermo-sensitive genic male sterility of PS001 and polyploid rice restorer line PR-T1 are used as experimental materials. They are used to anther and unfertilized ovary culture, and to get their diploid, respectively. [Result] In this paper, it creates a set of lines that contains diploid and tetraploid. The lines are photoperiod and thermo-sensitive genic male sterility and restorer line. [Conclusion] It is concluded that the anther and unfertilized ovary culture could be used to rapidly purify varieties and shorten the stable time of traits to achieve the purpose of rapid breeding, but also to create the appropriate materials for theoretical research.

Development of Commercial Thermo-sensitive Genic Male Sterile Rice Accelerates Hybrid Rice Breeding Using the CRISPR/Cas9-mediated TMS5 Editing System.

Hybrid rice breeding offers an important strategy to improve rice production, in which the cultivation of a male sterile line is the key to the success of cross-breeding. CRISPR/Cas9 systems have been widely used in target-site genome editing, whereas their application for crop genetic improvement has been rarely reported. Here, using the CRISPR/Cas9 system, we induced specific mutations in TMS5, which is the most widely applied thermo-sensitive genic male sterility (TGMS) gene in China, and developed new “transgene clean” TGMS lines. We designed 10 target sites in the coding region of TMS5 for targeted mutagenesis using the CRISPR/Cas9 system and assessed the potential rates of on- and off-target effects. Finally, we established the most efficient construct, the TMS5ab construct, for breeding potentially applicable “transgene clean” TGMS lines. We also discussed factors that affect the editing efficiency according to the characteristics of different target sequences. Notably, using the TMS5ab construct, we developed 11 new “transgene clean” TGMS lines with potential applications in hybrid breeding within only one year in both rice subspecies. The application of our system not only significantly accelerates the breeding of sterile lines but also facilitates the exploitation of heterosis.

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.

Comprehensive analyses of the annexin gene family in wheat.

BackgroundAnnexins are an evolutionarily conserved multigene family of calcium-dependent phospholipid binding proteins that play important roles in stress resistance and plant development. They have been relatively well characterized in model plants Arabidopsis (Arabidopsis thaliana) and rice (Oryza sativa), but nothing has been reported in hexaploid bread wheat (Triticum aestivum) and barely (Hordeum vulgare), which are the two most economically important plants.ResultsBased on available genomic and transcriptomic data, 25 and 11 putative annexin genes were found through in silico analysis in wheat and barley, respectively. Additionally, eight and 11 annexin genes were identified from the draft genome sequences of Triticum urartu and Aegilops tauschii, progenitor for the A and D genome of wheat, respectively. By phylogenetic analysis, annexins in these four species together with other monocots and eudicots were classified into six different orthologous groups. Pi values of each of Ann1–12 genes among T. aestivum, T. urartu, A. tauschii and H. vulgare species was very low, with the exception of Ann2 and Ann5 genes. Ann2 gene has been under positive selection, but Ann6 and Ann7 have been under purifying selection among the four species in their evolutionary histories. The nucleotide diversities of Ann1–12 genes in the four species were 0.52065, 0.59239, 0.60691 and 0.53421, respectively. No selective pressure was operated on annexin genes in the same species. Gene expression patterns obtained by real-time PCR and re-analyzing the public microarray data revealed differential temporal and spatial regulation of annexin genes in wheat under different abiotic stress conditions such as salinity, drought, cold and abscisic acid. Among those genes, TaAnn10 is specifically expressed in the anther but fails to be induced by low temperature in thermosensitive genic male sterile lines, suggesting that specific down-regulation of TaAnn10 is associated with conditional male sterility in wheat.ConclusionsThis study analyzed the size and composition of the annexin gene family in wheat and barley, and investigated differential tissue-specific and stress responsive expression profiles of the gene family in wheat. These results provided significant information for understanding the diverse roles of plant annexins and opened a new avenue for functional studies of cold induced male sterility in wheat.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-016-2750-y) contains supplementary material, which is available to authorized users.

Premature breakdown of tapetum associated with reverse thermo-sensitive genic male-sterile line Huiyou50S in rapeseed (Brassica napus)

Thermo-sensitive genic male-sterility (TGMS) has great advantages in hybrid crop production. Different from common TGMS in Brassica napus, a reverse TGMS line named Huiyou50S is male sterile when it is cultivated in low temperature, but highly fertile when exposed to high temperature. To gain further insights into the developmental aberrations leading to this male sterility, the anther development of Huiyou50S was observed by light and electron microscopy. Firstly, it was suggested by light microscopy that the abortion of anther occurred at the tetrad to early uninucleate microspore stage. A premature breakdown of tapetum is associated with protoplast degradation in the uninucleate microspores. Under transmission electron microscope, the tapetum exhibited vacuolization from pollen mother cell (PMC) stage to tetrad microspore stage and was then disrupted before late microspore stage. Alterations in the tapetal cells included abnormal plastids and a loss of recognizable elaioplast. However, the endoplasmic reticulum seemed to be normal as in the fertile plant. The formation of primexine on the surface of tetrad microspores was postponed to the uninucleate microspore, and hence exine construction was delayed. The evidences suggested that a premature breakdown of tapetum characterized by vacuolization, less secretory activity and abnormal elaioplast, rather than programmed cell death in normal plant, was associated with microspore abortion in Huiyou50S.

A comparative study of two-line early season hybrid rice with lepidopteran resistance

In our previous study, an early season rice restorer line B2A68 expressing Cry2Aa# gene was obtained. In order to evaluate the performance of B2A68’s hybrids, a total of six hybrids from crossing B2A68 with six thermo-sensitive genic male sterile (TGMS) lines of early season rice were compared for expression level of Cry2Aa protein, as well as in insect resistance, agronomic traits and grain quality with their non-Bt null controls. Our results showed that the highest Cry2Aa protein content among B2A68's hybrids was detected in leaf, where the Cry2Aa content was nearly three times of that in stem. The Cry2Aa content of B2A68 was significantly higher than those of its derived hybrids, indicating the Cry2Aa# gene was incompletely dominant in hybrids. The hybrids with Cry2Aa# gene exhibited higher plant protection against rice leaf roller than the hybrids without Cry2Aa# gene in field. Significant differences were observed in plant height, panicle length, number of grains per panicle, seed set, 1000-grain weight and yield per plant between hybrids with Cry2Aa# gene and hybrids without Cry2Aa# gene in 2013 or 2014. There were significant differences in chalky grain rate and chalkiness degree between paternal lines B2A68 and D68, and between hybrids with Cry2Aa# gene and hybrids without Cry2Aa# gene. All of the differences in agronomic traits and grain quality varied among parental lines and hybrids. In this paper, three hybrids with high lepidopteran resistance, high yield, elite agronomic traits and acceptable grain quality was selected out, that could serve as elite resistant hybrids to control lepidopteran pests in early season rice production.

Development of SNP-based dCAPS markers for identifying male sterile gene tms5 in two-line hybrid rice.

Molecular markers can increase both the efficiency and speed of breeding programs. Functional markers that detect the functional mutations causing phenotypic changes offer a precise method for genetic identification. In this study, we used newly derived cleaved amplified polymorphic sequence markers to detect the functional mutations of tms5, which is a male sterile gene that is widely used in rice production in China. In addition, restriction cutting sites were designed to specifically digest amplicons of tms5 but not wild type (TMS5), in order to avoid the risk of false positive results. By optimizing the condition of the polymerase chain reaction amplifications and restriction enzyme digestions, the newly designed markers could accurately distinguish between tms5 and TMS5. These markers can be applied in marker-assisted selection for breeding novel thermo-sensitive genic male sterile (TGMS) lines, as well as to rapidly identify the TGMS hybrid seed purity.

Development of broad‐spectrum bacterial blight resistance into thermo‐sensitive genic male sterile lines

AbstractTwo‐line hybrid rice technology is an effective way to increase rice production and improve rice quality. In this study, three bacterial blight (BB) resistance genes, Xa7, Xa21 and Xa23, were introgressed separately into C815S, a popular thermo‐sensitive genic male sterile (TGMS) line to develop five BB‐resistant lines (Hua1005S, Hua1002S, Hua1009S, Hua1006S and Hua1001S) to be resistant against seven races of Xanthomonas oryzae pv. oryzae (Xoo). The two‐line hybrids with heterozygous Xa23 were resistant against seven Xoo strains. But, the hybrids with heterozygous loci for both Xa7 and Xa21 were only resistant against three Xoo strains and were moderately susceptible to the other four strains indicating the role of modifiers influencing the poor expression of dominant BB resistance genes under heterozygous state. Among them, Hua1006S was found to be a promising TGMS line with its higher degree of disease resistance level on account of broad‐spectrum resistance gene Xa23 besides possessing better plant type and rice grain quality features.

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.

Molecular breeding of thermo-sensitive genic male sterile (TGMS) lines of rice for blast resistance using Pi2 gene.

BackgroundBlast disease caused by the fungal pathogen Magnaporthe oryzae is one of the big problems in rice production in China, especially for high yield hybrid varieties made from a two-line system in which thermo-sensitive genic male sterile (TGMS) lines are used. In this study, we report the introgression of a rice blast resistance gene Pi2 from VE6219 into C815S, an elite rice TGMS line, leading to the development of blast resistant TGMS lines through marker assisted selection (MAS) and phenotypic selection approaches.ResultsFour new TGMS lines with blast resistance gene Pi2 were developed from C815S (an elite TGMS line susceptible to the blast, used as recurrent parent) and VE6219 (a blast resistant line harboring Pi2, used as donor parent). The pathogenicity assays inoculated with 53 blast prevalent isolates in glasshouse showed that the blast resistant frequency of the four TGMS lines was 94.3%-98.1% that is equivalent to blast resistant donor parent VE6219. The field evaluation of the new lines and hybrids made from them at a blast epidemic site also showed high resistant levels against the blast. The genetic background of the newly developed TGMS lines were examined using a whole-genome single nucleotide polymorphism (SNP) array (RICE6K) that turned out more than 83% of the genomic markers were derived from the recurrent parent. The critical temperature points of fertility-sterility alteration of the new TGMS lines were between 22°C and 23°C of daily mean temperature, which is similar to that of C815S. The complete male sterility under natural growth conditions at Wuhan last more than 80 days. Their agronomic and grain quality traits meet the requirement for two-line hybrid rice production.ConclusionsThe broad-spectrum and durable rice blast resistant gene Pi2 was introgressed into the elite TGMS line C815S background. The newly developed TGMS lines can be practically used for two-line hybrid rice breeding and must play an important role in sustainable rice production in China.

The rice thermo-sensitive genic male sterility gene tms9: pollen abortion and gene isolation

AbstractThe development of two-line hybrid rice has done great contribution to the food security. It is imperative to study the male sterility mechanism of rice photo-thermo sensitive genic male sterile (PTGMS) line which is the core component of two-line hybrid rice. Zhu1S is a rice thermo-sensitive genic male sterile line used frequently as female parent in two-line hybrid seed production. A cytological examination of the development of the Zhu1S anther wall and the microspores showed that tms9 encoded male sterility is caused by the failure of the tapetum to degenerate normally, thereby starving the microspores and finally leading to pollen abortion. A fine-scale genetic map based on a large F2 population allowed tms9 to be located within a 30.2 Kb segment of chromosome 2 harboring seven open reading frames. A comparison between Zhu1S under sterile temperature condition (high temperature, male sterile) and fertile temperature condition (low temperature, fertile) showed that only one of the seven genes, LOC_Os02g12290, was differentially transcribed, with its transcript abundance being much lower under the sterile temperature growing condition. LOC_Os02g12290 encodes a nuclear ribonuclease Z. Re-sequencing demonstrated that the Zhu1S LOC_Os02g12290 allele differed from that present in non tms9 carrier alleles by two contiguous nucleotides in the first exon, inducing a truncated mRNA.

RNase Z(S1) processes UbL40 mRNAs and controls thermosensitive genic male sterility in rice.

Thermosensitive genic male-sterile (TGMS) lines, which are male-sterile at restrictive (high) temperatures but male-fertile at permissive (low) temperatures, have been widely used in breeding two-line hybrid rice (Oryza sativa L.). Here we find that mutation of thermosensitive genic male sterile 5 (tms5) in rice causes the TGMS trait through a loss of RNase Z(S1) function. We show that RNase Z(S1) processes the mRNAs of three ubiquitin fusion ribosomal protein L40 (UbL40) genes into multiple fragments in vitro and in vivo. In tms5 mutants, high temperature results in increased levels of UbL40 mRNAs. Overaccumulation of UbL40 mRNAs causes defective pollen production and male sterility. Our results uncover a novel mechanism of RNase Z(S1)-mediated UbL40 mRNA regulation and shows that loss of this regulation produces TGMS in rice, a finding with potential applications in hybrid crop breeding.

Genetic analysis and chromosome mapping of a thermo-sensitive genic male sterile gene in wheat

BS20-T is a thermo-sensitive genic male sterile (TGMS) wheat line (Triticum aestivum L.) used in hybrid wheat breeding in northern China. BS20-T was crossed with common wheat variety ZN7. F1 plants were highly fertile. The segregation ratio of male fertile and male sterile individuals in F2 and BC1F1 plants were close to 3:1 and 1:1, respectively. Other crosses of BS20-T also segregated for a single gene. Bulked sergeant analysis with SSR markers indicated the sterility gene was located on chromosome 2BL. It was named tmsBS20T. Screening of 162 extremely sterile and 30 extremely fertile F2 plants with linked markers indicated flanking by gwm403 and gwm374 with genetic distances of 2.2 and 4.5 cM, respectively. tmsBS20T was transferred into several common wheat varieties by using traditional hybridization combined with marker assisted selection with these two markers. Further identification of derived TGMS lines is ongoing.

Photosynthesis and Hill Reaction - a Physiological Inquiry into a Thermosensitive Genic Male Sterile (TGMS) Rice Used in Two Line Breeding

An investigation was carried out to understand the physiological mechanisms of male sterility in rice. The critical sterility temperature and critical stage of thermosensitivity of the selected TGMS (Thermosensitive Genie Male Sterile) line, TS 29 was studied using a Phytotron. Variations in the physiological traits were analyzed at Stage VII of panicle development (Filling phase of pollen, floret and panicle reach full length and colour turns to green). High significant reductions in photosynthetic rate, transpiration rate, hill reaction, sugars and carbohydrate contents were recorded. The high temperature induced male sterility in TGMS line was associated with accumulation of starch and higher diffusive resistance. However a reverse of all the above responses was found in the TS 29 grown under fertility inducing low temperature and in the variety CO 46. The results revealed that some physiological traits play an important role by acting as signals or messengers in response to temperature leading to pollen abortion.

Selection of Submergence Tolerant Homozygous Line by STS Marker and Twice Submergence Stress

One sequence tagged site marker Sub1-1 and twice submergence stress method were used in selection of submergence tolerant homozygous line from Sub-1BS, a submergence tolerant, bentazon sensitive and photoperiod-sensitive and/or thermo-sensitive genic male sterile line that developed by our laboratory. The results revealed that the original Sub-1BS was heterozygous in Sub1A-1 locus even though it was identical in almost all of agronomical traits and the segregation of Sub1A-1 was in accordance with Mendelian law based on chi-square test. And then the original Sub-1BS was divided into two groups: one was of Sub1A-1 introgression and the other was not; and the two groups were tested by twice submergence stress method. After the first submergence stress that lasted for 12 d, the average plant heights were significant difference at the 1% level between the two groups. After recovery for 10 d, the second submergence stress sustained for 18 d was carried on; and the group with Sub1A-1 gene was found apparently tolerant than the other group in submergence tolerance.

Sterility mechanism in different TGMS and CMS lines of rice

Thermosensitive genic male sterility (TGMS) is a new genetic tool used in developing commercial F 1 rice hybrids. This study investigated the stage at which sterility occurs during anther development in TGMS lines. For critical examination, panicles were collected from TGMS lines TS16, TS18, TS29, and cytoplasmic male sterile (CMS) lines IR58025A and IR62829A. In the TGMS lines, the developmental stages of young panicles were examined at stages IV, V, and VI in September 1998 and May 1999 for the expression of fertility and sterility, respectively. Anthers were taken out from the spikelet and fixed in 70% alcohol for 1 h. These were then passed through a series of solutions containing alcohol and methyl salicylate in the proportion of 4:1, 3:2, 2:3, and 1:4, and finally in methyl salicylate for 15 min in each solution with two changes in the last step. Cleared anthers were mounted in methyl salicylate in a cavity microslide and examined using a Namarski differential interference contrast in a bright field microscope for optimal viewing and microphotography.

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.