Expression of thermosensitive genic male sterility in rice under varying temperature situations

Response of thermosensitive genic male sterility in rice to varying temperature situations was studied by using four TGMS lines. In three sets of maximum, minimum and their combined temperatures, it was observed that maximum temperature played a predominant role in influencing sterility/fertility of TGMS lines under the combined regimes. Expression of a TGMS gene was found to be influenced by the genetic background of the recipient lines. Exposure for more than 8 hours of 32 ^°C was found necessary to induce complete male sterility in indica TGMS lines: IR68945-4-33-4-14 and IR68949-11-5-31 while, more than 4 hours of such exposure was enough to induce sterility in case of their japonica donor Norin PL 12. Sudden interruption with 27 ^°C even for 2 hours under the sterility inducing regime of 32/24 ^°C could induce partial fertility in the line IR68945-4-33-4-14. However, the line ID 24 remained completely sterile even with 10 hours of interruption with 27 ^°C. An interruption with lower temperature of 27 ^°C for 4 hours for even one day induced partial fertility in IR68945-4-33-4-14. The period of four to eight days after panicle initiation was the stage most sensitive to temperature variations. Hybrid rice breeders need to develop numerous genetically diverse TGMS lines, which possess critical sterility inducing temperature of 28 ^°C and are not affected by sudden interruptions with a lower temperature for few hours daily for a couple of days. This revised version was published online in August 2006 with corrections to the Cover Date.     

Interaction between the eui gene and thermo-sensitive genic male sterility in rice

Panicle enclosure is a typical phenotype of almost all male-sterile rice lines. An elongated uppermost internode (eui) mutant exhibited notably rapid elongation of the uppermost internode at the heading stage; this is considered as a potential mechanism to eliminate panicle enclosure. We developed thermo-sensitive genic male-sterile (TGMS) eui mutants that were characterized by notably elongated uppermost internodes. The elongation of the uppermost internode in the TGMS eui mutant Changxuan 3S (CX) is mainly attributed to an increase in cell number and cell elongation, the latter being the more significant process. Temperature treatments revealed that the effects of temperature on panicle exsertion were similar to those on fertility and that the most temperature-sensitive stage coincides with the period from the formation of the pollen mother cell to meiosis during panicle initiation. These results indicate that elongation increases as temperature decreases and that the expression of the eui gene is more efficient at low temperatures than at high temperatures. In hybrid rice seed production using the TGMS eui mutant, the temperature range should be optimized at 24–28°C in order to preserve the development of completely male-sterile pollen and to eliminate panicle enclosure. Consequently, by using TGMS eui rice lines, gibberellin application can be avoided, thereby reducing the cost of hybrid seed production. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Genetic analysis of male sterility in rice mutants with environmentally influenced levels of fertility

Summary Twenty-six male sterile plants grown in the field were recovered in the M7 generation from ethyl methane sulfonate-treated material of the rice cultivar M-201. Fertility increased five-fold when ratooned plants from the field were grown in a growth chamber with a 12 hour daylength. Crosses between mutant and normal fertile cultivars produced fertile F₁ plants. Female fertility was normal as judged by percent seed set from unbagged panicles of parental and recombinant lines. Transgressive segregation for fertility was observed for all crosses in the F₂ and F₃ generations. Five of 37 F₃ male sterile plants showed moderate levels of seed fertility under winter greenhouse conditions and reduced seed set when transplanted to summer field plots. Fertility data from reciprocal crosses suggested cytoplasmic factors had little or no effect on levels of male sterility in the mutant lines. Chi-squared analyses of F₂ and F₃ generation results indicated male sterility of the mutants is conditioned by two nuclear genes with epistatic effects.     

Combining ability and heterosis over environments for yield and yield components in two-line hybrids involving thermosensitive genic male sterile lines in rice (Oryza sativa L.)

Presence of substantial heterosis and economic hybrid seed production are two most desirable components for success of any commercial hybrid breeding programme. Thermosensitive genic male sterile (TGMS) lines of rice, in this regard, have tremendous potential in realizing further quantum jump in yield and economical hybrid seed cost. Analyses for combining ability and heterosis over optimum (120N : 60P₂O₅ : 40K₂O kg/ha) and high (200N : 90P₂O₅ : 60K₂O kg/ha) fertility environments for six traits were made in 2 years (2001 and 2002) using 120 hybrids of inter‐ and intra‐subspecific nature derived from hybridization of 30 elite indica TGMS lines and four cultivars, viz., ‘Pant Dhan 4’ and ‘Ajaya’ (I = indica), ‘Taichung 65’ (J = japonica) and ‘IR 65598‐112‐2’ (TJ = tropical japonica) in line × tester mating design. Predominance of non‐additive genetic variance suggested good prospects of hybrid breeding. Pooled analysis revealed highly significant variances for lines, general combining ability (GCA), specific combining ability (SCA) and line x tester. TGMS line 365‐8S was the best general combiner for all the six traits including grain yield. Trend of relative mid‐parent heterosis for grain yield, panicle length, grain number per panicle and earliness in flowering was I/TJ > I/J > I/I. For panicle number per plant and 1000‐grain weight, trends were I/TJ > I/I > I/J and I/I > I/TJ > I/J, respectively. Grain yield recorded heterosis of 49.3%, 71.9% and 92.7% for I/I, I/J and I/TJ hybrid groups respectively. Effect of environments on the hybrid performance indicated better response of hybrids at high fertilizer dose. Study suggests greater prospects of combining improved japonica and tropical japonica germplasms having wide compatible gene with indica TGMS lines for exploitation of intersubspecific heterosis.     

A novel environment-induced genic male sterile (EGMS) mutant in indica rice

Summary Male-sterile mutants were isolated from M₂ and M₃ generations of indica rice variety 26 Zhaizao, dry seeds of which had been exposed to ⁶⁰Co- rays at a dose of 290 Gy. The mutants were planted in early season and ratooned in late season for two successive years for identification of fertility conversion in different growing seasons. One of the mutant lines was further observed in a growth chamber and in the field. Results showed that daily average temperature might be the major factor conditioning the male fertility conversion at a moderate daylength. The critical temperature for the male fertility conversion of the mutant grown under 12.5 h and 14.0 h daylength is about 23°C, below which the plant becomes completely male sterile. Its male fertility conversion character differs from other EGMS lines so far developed. The performance of the hybrids between the mutant and some other indica varieties demonstrated its good combining ability and its potential value in hybrid rice production. The obtained mutant line still sheds KI-stainable pollen grains under male sterilizing conditions. Nevertheless, pollen grains shed from the male sterile plants were much more vulnerable than from normal plants. At sucrose concentration below 1.5 M, the pollen grains from the mutant grown under male sterilizing conditions almost completely broke down, while above 1.5 M they became plasmolysed and shrunken. This is indicative of poor development of the membrane and walls of the pollen grains from the male sterile mutant, causing the pollen grains to be unfunctional. NBT test also clarified the abortion of the pollen grains from the mutant, which were formed in the male sterilizing environment.     

The wi gene causes genic male sterility in Allium choenoprasum

Segregation studies following the transfer of the gene wi to different cytoplasm types, which have been distinguished by means of restriction fragment length polymorphism analyses using mitochondrial gene probes, revealed the formation of the wi‐sterility in each of the four cytoplasms examined. The male sterility is therefore only caused by the nuclear wi gene, i.e. an additional factor of a specific cytoplasm can be excluded. Hence, the wi‐sterility proved to be a genic male sterility (GMS) and not a cytoplasmic male sterility (CMS). The expression of the wi‐sterility appears to be stable, since it is not affected by high temperatures or tetracycline. Accordingly, a temporary pollen production, which would allow self‐fertilization for the maintenance of sterile lines, cannot be induced by controlling these environmental factors. In terms of hybrid breeding, this GMS therefore has no advantage over the previously described CMS system.     

Development of TGMS lines for developing two-line rice hybrids for the tropics

A breeding program was initiated at the International Rice Research Institute (IRRI) in 1990 to develop thermosensitive genic male sterile (TGMS) rice lines for developing two-line rice hybrids for the tropics. The TGMS trait was transferred from a temperate japonica TGMS mutant, Norin PL 12, to indica and tropical japonica rice varieties using the pedigree selection procedure. Six new TGMS rice lines adapted to tropical conditions were developed which showed complete pollen and spikelet sterility when maximum temperature was higher than 30 ^°C 1–2 week after panicle initiation. However, up to 85.5% spikelet fertility was observed when these lines were exposed to 26–29 ^°C during the critical stage. Using two of these TGMS lines, some heterotic rice hybrids showing 1–1.6 t/ha higher grain yield than the inbred check varieties were identified in unreplicated observational yield trial conducted at IRRI. Two of the six two-line hybrids yielded significantly higher than the check variety in a replicated preliminary yield trial. This revised version was published online in July 2006 with corrections to the Cover Date.     

Chromosomal location of fertility restoring genes for ‘wild abortive’ cytoplasmic male sterility using primary trisomics in rice

Summary Identification and location of fertility restoring genes facilitates their deployment in a hybrid breeding program involving cytoplasmic male sterility (CMS) system. The study aimed to locate fertility restorer genes of CMSWA system on specific chromosomes of rice using primary trisomics of IR36 (restorer), CMS (IR58025A) and maintainer (IR58025B) lines. Primary trisomic series (Triplo 1 to 12) was crossed as maternal parent with the maintainer line IR58025B. The selected trisomic and disomic F₁ plants were testcrossed as male parents with the CMS line IR58025A. Plants in testcross families derived from disomic F₁ plants (Group I crosses) were all diploid; however, in the testcross families derived from trisomic F₁ plants (Group II crosses), some trisomic plants were observed. Diploid plants in all testcross families were analyzed for pollen fertility using 1% IKI stain. All testeross families from Group I crosses segregated in the ratio of 2 fertile: 1 partially fertile+partially sterile: 1 sterile plants indicating that fertility restoration was controlled by two independent dominant genes: one of the genes was stronger than the other. Testcross families from Group II crosses segregated in 2 fertile: 1 partially fertile+ partially sterile: 1 sterile plants in crosses involving Triplo 1, 4, 5, 6, 8, 9, 11 and 12, but families involving triplo 7 and triplo 10 showed significantly higher X² values, indicating that the two fertility restorer genes were located on chromosome 7 and 10. Stronger restorer gene (Rf-WA-1) was located on chromosome 7 and weaker restorer gene (Rf-WA-2) was located on chromosome 10. These findings should facilitate tagging of these genes with molecular markers with the ultimate aim to practice marker-aided selection for fertility restoration ability.     

Production and testing of plants regenerated from protoplasts of photoperiod sensitive genic male sterile rice (Oryza sativa L.)

Plants were regenerated from protoplasts isolated from embryonic suspension cultures of N5047S, a photoperiod sensitive genic male sterile (PGMS) Japonica rice line. Flow cytometric analyses of nuclear DNA content identified some tetraploid regenerates whose agronomic traits could be distinguished from diploid regenerates. Pollen and female fertility of diploid protoplast-derived clones grown under different light and temperature conditions was compared. A promising PGMS protoplast clone, ZAU11S, was developed from these clones. Its male sterility was confirmed as a photoperiod × temperature interaction type. This revised version was published online in July 2006 with corrections to the Cover Date.     

Analysis of genic male sterility in Brassica oleracea

Summary The male sterility system MS-1 of Brassica oleracea was studied in order to elucidate if nucleo-cytoplasmic interactions determine this system. Crosses of male sterile MS-1 genotypes with heterozygous MS-5 genotypes gave fully fertile F₁ progenies. Selfing of seven F₁ plants resulted in five F₂ populations showing a 9:7 segregation ratio and two a 3:1 ratio for fertile and male sterile plants. Two F₂ progenies deviated from the expected 9:7 or 3:1 segregation ratios for fertile and male sterile plants. Thermosensitivity and distortion of the meiosis are suggested as the causal factors underlying the deviation of the segregation ratios. It was concluded that nuclear factors determine the male sterility in the MS-1 system, because the presence of a nucleocytoplasmic interaction in this system should have given only a 3:1 segregation ratio for fertile and male sterile plants in the F₂ generation.     

Molecular mapping of S-c, an F1 pollen sterility gene in cultivated rice

Hybrids between indica and japonica rice varieties usually show partial sterility, and are a major limiting factor in the utilization of heterosis at subspecific level. When studying male-gamete (pollen) abortion, a possibly important cause for sterility, six loci (S-a, S-b, S-c, S-d, S-e and S-f) for F₁ pollen sterility were identified. Here we report genetic and linkage analysis of S-c locus using molecular markers in a cross between Taichung 65, a japonica variety carrying allele S-c ^j, and its isogenic line TISL5, carrying alleleS-c ^j. Our results show that pollen sterility occurring in the hybrids is controlled by one locus. We used 208 RFLP markers, as well as 500 RAPD primers, to survey the polymorphism between Taichung 65 and TISL5. Six RFLP markers located on a small region of chromosome 3, detected different RFLP patterns. Co-segregation analysis of fertility and RFLP patterns with 123 F₂ plants confirmed that the markers RG227, RG391, R1420 were completely linked with the S-c locus. The genetic distances between the markers C730, RG166 and RG369 and the S-c locus were 0.5 cM, 3.4 cM, and 3.4 cM respectively. Distorted F₂ ratios were also observed for these 4 RFLP markers in the cross. This result suggests that the `one locus sporo-gametophytic' model could explain F₁ hybrid pollen sterility in cultivated rice. RG227, the completely linked marker, has been converted to STS marker for marker-assisted selection. This revised version was published online in August 2006 with corrections to the Cover Date.     

Identification and transfer of a new cytoplasmic male sterility source from Oryza perennis into indica rice (O. sativa)

Summary Most of the commercial hybrids of indica rice are based on wild abortive (WA) source of cytoplasmic-genetic male sterility (CMS). Such cytoplasmic uniformity may lead to genetic vulnerability to disease and insect pests. To overcome this problem, diversification of CMS sources is essential. Crosses of 46 accessions of O. perennis and two accessions of O. rufipogon as female parents were made with two restorers (IR54, IR64) of WA cytosterility. Sterile hybrids were backcrossed with the respective recurrent parents. Of all the backcross derivatives, one line having the cytoplasm of O. perennis Acc 104823 and the nuclear background of IR64 was found to be stable for male sterility. The newly developed CMS line has been designated as IR66707A. This line is completely sterile (0% seed set) under selfed conditions. Crosses of IR66707A with 10 restorers of WA cytoplasm showed almost complete (93–100%) pollen sterility, indicating that the male sterility source of IR66707A is different from WA sterility. Southern hybridization of IR66707A, O. perennis (cytoplasmic donor), IR66707B (maintainer) and V20A (WA cytoplasm) using mitochondrial DNA specific probes (5 endonucleases × 8 probes) showed identical banding patterns between IR66707A and O. perennis. However, in more than half of the combinations, different banding patterns were observed between IR66707A and IR66707B and between IR66707A and V20A. The results suggest that IR66707A has the same cytoplasm as the donor (O. perennis), and CMS may not be caused by any major rearrangement or modification of mtDNA. The new CMS source identified will be useful in cytoplasmic diversification in hybrid rice breeding.     

Identification and characterization of new sources of cytoplasmic male sterility in rice

Search for male sterility-inducing cytoplasm in wild species of the genus Oryza was attempted with a view to diversify the base of the cytoplasmic genetic male sterility system currently used in the development of commercial rice hybrids. A total of 132 interspecific crosses were made involving accessions of four wild and two cultivated species, all belonging to the A genome. Wild accessions possessing sterility-inducing cytoplasms were identified following reciprocal and sterile F₂ backcross methods. Sterile segregants were pursued through substitution backcrosses to develop cytoplasmic male-sterile (CMS) lines. CMS lines were developed with the cytoplasm of either O. rufipogon (VNI) or O. nivara (DRW 21018, DRW 21001, DRW 21039, DRW 21030 and RPW 21111). Based on shape, staining, and abortive pattern of pollen and also on type of interaction with a set of restorers and maintainers for known cytoplasmic male steriles of WA source (V 20A), the newly-developed CMS lines were grouped into four classes. Of these, RPMS 1 and RPMS 2 showed gametophytic male sterility with a restorer reaction different from WA cytoplasmic male-sterile stocks.     

Isolation and genetic characterization of a fertility-restoring revertant induced from cytoplasmic male sterile rice

Summary A male fertile revertant was isolated from M₁ of a cytoplasmic male sterile indica rice line II-32A, the dry seeds of which were treated with ⁶⁰Co- rays at a dose of 290 Gy. The acquired revertant T24 was morphologically and agronomically similar to II-32B, the maintainer of II-32A. Testcrosses of the revertant with II-32A and Zhenshan 97A showed that the revertant was able to restore the fertility of CMS lines. Genetic analysis of the progenies between T24 and II-32A, Zhenshan 97A XieqingzaoA and DZhenshan 97A, which have different cytoplasms, showed that the fertility restoration of four CMS lines by T24 involved one nuclear gene, indicating that T24 was a result of the mutation of one nuclear gene. The mechanism of the restoration of CMS line by T24 was obviously different from other restorers such as Minghui 63 and 20964, which were shown to behave in two gene mode in fertility restoration. The discovery of the revertant T24 contributes to the understanding of CMS and fertility restoration of CMS in rice. The T24 and its parent II-32A may constitute a pair of near isogenic lines for the restoring gene, which should be valuable materials for molecular genetic analysis of CMS.     

Cytological observations in some hybrids between the rice species Oryza sativa L. and O. glaberrima Steud.

Summary Several hybrids between Oryza sativa and O. glaberrima and their backcrosses with O. sativa were studied. Their seed sterility was very different; large differences were also observed in the level of pollen sterility and in the earliness of microspore failure. The proportion of aborted embryo sacs was much lower than the rate of sterile male gametophytes. The backcross populations were much more sterile than the corresponding F₁ hybrids. On the base of our observations and according to the literature, we may conclude that genic unbalance is the main cause of sterility of these hybrids, but that physiological factors may also be involved. Thus a restoration of fertility is generally possible by selection. On the other hand, male-sterile lines could be bred from some of these hybrids.     

Inheritance of photoperiod sensitive genic male sterility and breeding of photoperiod sensitive genic male-sterile lines in rice (Oryza sativa L.) through anther culture

The fertility segregations of F₁, F₂, BCF₁ descended from crosses between PSGMR and japonica varieties, and F₁'s anther cultured homozygous diploid pollen plant populations (H₂) were studied to reveal the genetic mechanism of photoperiod sensitive genic male sterility in PSGMR under natural daylight length at Shanghai. Rate of bagged seed-setting was used as an indicator of fertility. Fifteen F₁ showed complete fertility similar to their parents. The ratio of completely sterile plants to fertile plants in fifteen F₂ and four BCF₁ was 1:15 and 1:3, respectively. The ratio of completely sterile to fertile diploid pollen plants in nine diploid populations (H₂) was 1:3. These results demonstrated that the photoperiod sensitive genic male sterility in PSGMR was governed by two pairs of independent major recessive genes. There were no significant fertility segregations in hybrids F₁ and selfed F₂ between Nongken 58S and its derivatives 7001S, 5088S, 5047S and M105-9S, indicating that the photoperiod sensitive genic male-sterile genes in Nongken 58S were allelic to those in its derivatives. Several photoperiod sensitive genic male-sterile diploid pollen lines were bred from anther cultured homozygous diploid populations (H₂) in about a three-year period. Most of these diploid lines showed significant fertility transformation and stable complete sterility from 5 August to 5 September, excellent agronomic traits and high resistance to blast and bacterial leaf blight. This revised version was published online in July 2006 with corrections to the Cover Date.     

Development of photoperiod- and thermo-sensitive male sterility rice expressing transgene Bacillus thuringiensis

Stem borers and leaffolders are the main pests that cause severe damage in rice (Oryza sativa L.) production worldwide. We developed the first photoperiod- and thermo-sensitive male sterility (PTSMS) rice 208S with the cry1Ab/1Ac Bacillus thuringiensis (Bt) gene, through sexual crossing with Huahui 1 (elite line with the cry1Ab/1Ac gene). The novel 208S and its hybrids presented high and stable resistance to stem borers and leaffolders, and the content of Cry1Ab/1Ac protein in chlorophyllous tissues achieved the identical level as donor and showed little accumulation in non-chlorophyllous tissue. No dominant dosage effect in the Bt gene was observed in 208S and its derived hybrids. An analysis of fertility transition traits indicated that 208S was completely sterile under long day length/high temperature, but partially fertile under short day length/low temperature. With fine grain quality and favorable combining ability, 208S had no observed negative effects on fertility and agronomic traits from Bt (cry1Ab/1Ac). Additionally, 208S as a male sterile line showed no fertility decrease caused by Bt transgenic process, as it is the case in Huahui 1. Thus, 208S has great application value in two-line hybrid production for insect resistance, and can also be used as a bridge material in rice Bt transgenic breeding.     

Pyramiding of thermosensitive genetic male sterility (TGMS) genes and identification of a candidate tms5 gene in rice

Three thermosensitive genetic male sterility (TGMS) genes – tms2, tgms and tms5 – were pyramided using linked microsatellite markers. Three TGMS donors, Norin PL 12 (tms2), SA2 (tgms) and DQ200047-21 (tms5) were utilized in generating crosses from which two-gene and three-gene pyramids possessing the RM11 allele of Norin PL 12, RM257 allele of SA2 and RM174 allele of DQ200047-21 were selected. All selected progenies were male-sterile at sterility-inducing conditions. In addition, rice SF21 was identified as a candidate tms5 gene because of its complete linkage with RM174. The 4,200-bp region was amplified from the TGMS line M105S and the two ends were sequenced. In silico analysis of partial nucleotide sequences showed that the region is similar to the SF21 pollen-specific gene of Arabidopsis and Helianthus. The M105S tms5 sequence was also compared to the SF21 sequence from the International Rice Genome Sequencing Project (IRGSP) database.     

Cytoplasmically controlled cold tolerance in a cytoplasmic-genetic male sterile line of rice

Summary The cytoplasmic-genetic male sterile line, Krishna-A with Kalinga-I cytoplasm was developed in rice through repeated backcrossing the completely pollen sterile F₁ of the cross Kalinga-I/Krishna with the recurrent male parent cv. Krishna. The germination percentage and root-shoot length in Krishna-A at 12°C and 9°C indicated a higher degree of tolerance to low temperature than the cold tolerant female parent Kalinga-I. The high yielding cold tolerant variety Kalinga-I was developed from a cross involving the cold tolerant variety Dunghansali and the high yielding variety IR 8. It is inferred that tolerance to low temperature during germination in Krishna-A was inherited from the cytoplasm of cv. Dunghansali through Kalinga-I besides male sterility. The cytoplasmic control of cold tolerance is reported here for the first time in rice literature. The male sterile line Krishna-A because of its tolerance to low temperature would be suitable for the development of hybrid rice especially for areas where low temperature is a problem during germination.     

Use of herbicide-resistant genic male sterility in hybrid rice seed production

One of the difficult problems in hybrid rice seed production is the low outcrossing frequency and requirement for much labor to produce hybrid seeds. In order to simplify the process of hybrid rice seed production, herbicide-resistant photoperiod sensitive genic male sterile (HRPGMS) rice was utilized in this study. The herbicide resistance gene bar was transferred into the photoperiod sensitive genic male sterile (PGMS) rice 920S by Agrobacterium-mediated transformation and the HRPGMS line YA3530ms with good agronomic characteristics was bred by applying conventional pedigree breeding technique. The seeds of HRPGMS and pollen parent were mixed with the ratio of 4:1 in weight, and were sowed in seedling box. The mixed seedlings of HRPGMS and pollen parent grown for 30 days were transplanted by the small transplanting machine in the field. The herbicide glufosinate ammonium was sprayed at 7 days after flowering to kill all the plants of pollen parent, whereas hybrid seeds were harvested from the survived HRPGMS parent at maturity. The outcrossing frequency of HRPGMS line from two combinations in 2002 and from five combinations in 2004 were compared with a control cultivated by the conventional 2-line system. As the result, the mean outcrossing frequency in HRPGMS of the treatments were 10.6–24.5% compared with 5.5% in PGMS of the control in 2002, and that were 24.7–32.0% compared with 7.5% in the control in 2004. Consequently, using HRPGMS in two-line system was proved to be a new method that would simplify the process of hybrid rice seed production and to increase outcrossing frequency without any artificial supplementary pollination processes.