Oriented Generation of Novel Thermo-Sensitive Genic Male Sterile Lines with Improved Grain Shape and Outcrossing Rate in Early-Season Rice

Two-line hybrid rice with excellent quality is preferred in the Chinese market.However,there is a trade-off between reducing costs for hybrid seed production and lowering the outcrossing rate of the sterile line,which is largely determined by the stigma exsertion rate(SER).In this study,we constructed mutants of male sterility lines with improved grain length(GL)and SER in three elite early-season indica rice varieties through targeted manipulation of the TMS5 and GS3 genes using CRISPR/Cas9-mediated multiplex systems.We obtained a series of marker-free gs3 single mutants and gs3tms5 double mutants with significantly higher SER,longer grains,and increased 1000-grain weight compared with the wild type(WT).Importantly,the typically thermo-sensitive genic male sterile(TGMS)trait with a higher SER was observed in gs3tms5 mutants,and their F1 hybrids exhibited remarkable improvements in grain shape and yield-related traits.Our findings provided an efficient method to generate new valuable TGMS germplasm with improved SER through the mutagenesis of GS3 and TMS5 synergistically,and demonstrated that GS3 had pleiotropic effects on grain size,SER,and grain quality in early-season indica rice.

Rice AGL1 determines grain size and sterile lemma identity

The grass spikelet is a unique inflorescence structure that determines grain size.Although many genetic factors have been well characterized for grain size and glume development,the underlying molecular mechanisms in rice are far from established.Here,we isolated rice gene,AGL1 that controlled grain size and determines the fate of the sterile lemma.Loss of function of AGL1 produced larger grains and reduced the size of the sterile lemma.Larger grains in the agl1 mutant were caused by a larger number of cells that were longer and wider than in the wild type.The sterile lemma in the mutant spikelet was converted to a rudimentary glume-like organ.Our findings showed that the AGL1(also named LAX1)protein positively regulated G1 expression,and negatively regulated NSG1 expression,thereby affecting the fate of the sterile lemma.Taken together,our results revealed that AGL1 played a key role in negative regulation of grain size by controlling cell proliferation and expansion,and supported the opinion that rudimentary glume and sterile lemma in rice are homologous organs.

Influential Factors of Restorer of New Cytoplasmic Male Sterile(NER) on Anther Culture

Some influential factors of anther culture were studied preliminarily by conducting anther culture of the restorers of new cytoplasmic male sterile （NER）. Several results were obtain from this experiment and they were listed as follow：① MS cultrure medium with such hormones as 2,4-D 2 mg/L,6-BA 0.5 mg/L, NAA 0.5 mg/L was the best suitable for callus induction of NER. ②The difference of induction rate was significantly different between different plant age groups. From the 110th day to 141th day,the induction rate was increased with the increase of age and the difference of induction rate reached 0.01 significant difference level. The induction rate reached the highest value in the 141th day then it declined gradually. ③The combined use of 2, 4-D and 6-BA with proper increase of 2,4-D was good for inducing callus. ④The green plantlet induction rate of NER was increased when the concentration of 6-BA increased from 2 mg/L to 4 mg/L. Adding ZT from 0.5 mg/L to 2 mg/L. 6-BA would led 2.47% increase of green plantlet olantlet induction rate.

Genetic Analysis and Mapping of Genes Involved in Fertility of Pingxiang Dominant Genic Male Sterile Rice

Pingxiang-dominant genic male sterile rice （PDGMSR） was the first dominant genic male sterile mutant identified in rice （Oryza sativa L.）, and the corresponding dominant genic male sterile gene was designated as Ms-p. The fertility of PDGMSR can be restored by introduction of a dominant epistatic fertility restoring gene in some rice varieties. In the present study, E823, an indica inbred rice variety, restored the fertility of PDGMSR, and the genetic pattern was found to be consistent with a dominant epistatic model, therefore, the dominant epistatic fertility restorer gene was designated as Rfe. The F2 population from the cross of PDGMSR/E823 was developed to map gene Rfe. The F2 plants with the genotypes Ms-pMs-pRferfe or Ms-pms-pRferfe were used to construct a fertile pool, and the corresponding sterile plants with genotypes Ms-pMs-prferfe or Ms-pms-prferfe were used to con- struct a sterile pool. The fertility restoring gene Rfe was mapped to one side of the microsatellite markers RM311 and RM3152 on rice chromosome 10, with genetic distances of 7.9 cM and 3.6 cM, respectively. The microsatellite markers around the location of the Ms-p gene were used to finely map the Ms-p gene. The findings of this study indicated that the microsatellite markers RM171 and RM6745 flanked the Ms-p gene, and the distances were 0.3 cM and 3.0 cM, respectively. On the basis of the sequence of rice chromosome 10, the physical distance between the two markers is approximately 730 kb. These findings facilitates molecular marker-assisted selection （MAS） of genes Ms-p and Rfe in rice breeding programs, and cloning them in the future.

Observation on Flowering Habit of Reverse Genic Sterile Rice FHS

[Objective] This study was to use reverse genic male sterile FHS better in field production. [Method] Low energy nitrogen ion beam was taken as a mutation source to conduct mutagenic treatment for indica rice 99-02 and the mutant FHS with special fertility that was isolated from their offspring. Some characteristics such as flowering habit and stigma exsertion rate of FHS were observed in this experiment. [Result] The reverse genic sterile rice FHS had an obvious peak flowering stage from 10:00 to 10:30, while the second peak flowering was from August 5 to August 10. Compared with PA64S, FHS flowered early and its flowering time was concentrated, showing that it is for seed propagation. The stigma exsertion rate of FHS was 85.8% and low exsertion rate was good for the purity of seed. [Conclusion] The reverse genic sterile rice FHS had good value in use, besides, it could also be used as comparison material for studying fertility alternation mechanism of photoperiod sensitive genic male sterile rice.

Heterosis Analysis and Utilization of Agronomic Traits of Rice Male Sterile Line Jiazhe 91A

[Objective]Provide a theoretical basis for the popularization and application of Jiazhe 91A through the research and analysis on the advantages and characteristics of F1 generation of Jiazhe 91A combinations. [Method]The mid-parent heterosis,heterobeltiosis,competitive advantage and average dominance of the F1 generation of the three combinations configured by Jiazhe 91A were analyzed by the comparison and appraisal test of combinations configured by Jiazhe 91A. [Result]the panicle shape of the F1 generation of the three combinations configured by Jiazhe 91A was larger,and the panicles number was greater than its parents,the growing period was shorter than that of Shanyou 63,while the spikelets per panicle,seed setting rate and yield were higher than the control Shanyou 63,and the yield was 2.7% to 12.1% higher than Shanyou 63. [Conclusion]The sterility of Jiazhe 91A sterility was stable,and had a strong restoring ability,so it had widespread application prospects.

Microtubule Structure and Male Sterility in a Gene-Cytoplasmic Male Sterile Line of Rice, Zhen Shan 97A

Histological changes that occur during microsporogenesis are documented in a gene-cytoplasmic male sterile rice ( Oryza saliva L.) line, Zhen Shan 97A, its maintainer line, Zhen Shan 97B, and the restorer line, Ce64 of a Mine hybrid rice production system. In the restorer line, Ce64, the developing microsporocytes have dense cytoplasm and a distinct set of circumferential microtubules around the nucleus. Successive cytokinesis results in the formation of tetrads. The microtubules within the cells of tetrads and microspores radiate from the surface of the nucleus towards the outer edge of the cytoplasm. Subsequent pollen development is normal. During the course of microspore formation tubulin speckles can be found in the cytoplasm. The general pattern of development and microtubule organization in the maintainer lined Zhen Shan 97B, is similar to Ce64, except that a few more tubulin speckles appear during microspore formation. In the case of the mate sterile line, Zhen Shan 97A, a number of abnormalities can be discerned during early microsporogenesis. These include vacuoles forming within the developing microsporocyte and faintly stained microtubules with no defined distribution pattern. Prominent tubulin speckles are common within the cytoplasm. For those microsporocytes that undergo meiosis, no defined organizational patterns of microtubules can be found within the tetrad. All microspores abort soon after. Abnormalities and defects in microtubule organization observed in Zhen Shan 97A showed that complex interactions between the cytoplasm and the nucleus began at very early stage of microsporocyte development.

Immunohistochemical Analysis of IAA in Anthers of the Photoperiod-sensitive Genic Male-sterile Rice

The immunohistochemical localization of IAA and the comparison of their relative levels were carried out for the first time in the anthers of Nongken 58S and its wild type Nongken 58 (Oryza sativa subsp. japonica) after long_day and short_day treatments. The distribution of free_IAA in anthers and its dynamic variation could be reflected by this method. The results showed that the IAA level in the anthers of Nongken 58S after long_day treatment was much lower than that in short_day_treated Nongken 58S and those in wild type Nongken 58 in five stages from pistil and stamen primordia formation to late uninucleate stage. The possible reasons for IAA deficiency in Nongken 58S_LD anthers and its relationship with fertility alteration were also discussed.

Preliminary Studies on the Effect of Photoperiod and Temperature on Male Fertility of a Cytoplasmic Male Sterile Line in Wheat

[Objective] The study aimed to reveal the effect of photoperiod and temperature on male fertility of cytoplasmic male sterile line Vtai911289a in wheat and discuss the mechanism of male fertility alteration. [ Method ] The sowing-date tests and designed conditions were conducted during 2003 -2005. [ Result] Fertility of Vtai911289a, could alter under specific photoperiod and temperature conditions. Temperature is one of the main factors influencing male fertility of the male sterile lines. Vtai911289a, showed stable sterility under the condition of the mean of daily temperature at fertility sensitive stage lower than 19℃ and presented partial fertility when the mean of daily temperature at fertility sensitive stage lower than 20 - 22℃. Photoperiod to some extent affects the male fertility of Vtai911289a, long-day condition is helpful for the male fertility of the sterile line. [ Conclusion] The application of photoperiod temperature-sensitive cytoplasmic male sterile line in production has a higher safety than that of temperature sensitive sterile line.

Invention and Utilization of Glutinous Sorghum Male Sterile Line 45A with High Starch and High Combining Ability

45A is a glutinous sorghum male sterile line with high starch and high combining ability bred by Rice and Sorghum Institute of Sichuan Academy of Agricultural Sciences in 1998, it is a coeno-species taking non-glutinous maintainer line TL169-239B which bred by Tieling Institute of Agricultural Sciences in 1991 as the female parent and glutinous maintainer line72B bred by the authors＇ institute as the male parent, then, based on the backcross breeding between glutinous single plant chosen from F2 segregation population and Tx623A. There are ten hybrid sorghum varieties which already have been examined and approved by national and above provincial（municipal） level units; the patent of this breeding method has been authorized （the patent number： ZL 2012 1 0129155.6）; 45A is protected by the Right of New Varieties of Plants, MOA, P.R. China （the variety right number： CNA20090576.1）. In this paper, the breeding process of 45A and the characteristics of 45A sterile line and its hybrid sorghum were summed up, what＇s more, the technical key points of high-yielding breed of 45A and the production of hybrid sorghum seeds were introduced, to provide data for further popularizing the sterile line.

Directional Breeding of An Oval-ecotype Male Sterile Line of Chinese Cabbage(Brassica campestris L. ssp. pekinensis)

[Objective] The study aimed to explore the method for directional breeding of a male-sterile line in oval-ecotype Chinese cabbage. [Method] Based on ＂Multiple Allele Hypothesis of Genic Male Sterile Chinese Cabbage＂, an inbred line ＇06048＇ of oval ecotype was used as the receptor, and male fertile plant of ＇AB12＇ was used as the donor line. Crossing, backcross, selfing, testcross and sibling were ap- plied to transfer the multiple alleles under the directional genetic model. [Result] Segregation ratio of every generation was consistent with theoretical value. A new male sterile line with 100% male sterility and ＇06048＇ horticultural traits was ob- tained successfully, which accomplished the transfer of male sterile multiple allele and horticultural characters of receptor line at the same time. [Conclusion] The re- search verifies that the model of directional transfer is feasible, provides a theoreti- cal basis for the directional transfer of Chinese cabbage with other horticultural traits whose genotype is msms. The model can also be applied to other Brassica crops, to generate genetic male sterile lines with specific botanical traits and high-quality economic traits.

Study on Mutagenic Effect of Space Flight in Two-line Sterile Rice

Seeds of two-line sterile rice cultivars Zhu 1S and Lu 18S were carried into space by ＂Shijian 8＂ breeding recoverable satellite, then planted in ground. Mutagenic effects from space induction were compared with those from y-irradiation and complex processing of space induction and y-irradiation. The results showed that agronomic effect was stimulated in Mo progenies of the two-line sterile rice varieties treated by space flight, and their radiosensitivities to the irradiation of space flight performed non-sensitive. The order of mutation frequency was determined to be SP ＋ γ 〉 γ 〉 SP in M2 generation. And a series of mutated elites（individuals） were screened. Physiological indices of mutants screened like the activity of protective enzymes were measured to explore the physiological and biochemical basis of biological effect in space environment to two-line sterile rice. The results of this study show that space mutation breeding is an effective novel mean for breeding.

The Relation between Anther's Nutrient Metabolism and Pollen Abortion of Male Sterile Lines in Lycium barbarum L.

[ Objective] This study deals with the relation between anther nutrient metabolism and pollen abortion of male sterile lines in Lycium barbarum L., and provides some theoretical references for the pollen abortion mechanism of male sterile lines in Lyciurn barbarurn L.. [Method] By using semi-thin section and cytochemistry technology, the accumulation and distribution of anther nutrient of male sterile and fertile lines in Lycium barbarum L. were observed and compared. [ Result] The result showed that after meiosis, starch grains in the connective parenchyma of anther decreased sharply, and starch grains in epidermis and endothecium were also greatly decreased, while nearly no lipid accumulated in tapetal cells in male sterile lines compared with the fertile lines. The tapetal cells and tetraspore had vacuoles appear successively, and then entered the degenerate process. During the degeneration stage of tapetal cells and tetraspore, starch grains in epidermis and endothecium were still in a smell number and in the connective parenchyme as well. [ Conclusion] The decrease of polysaccharide supplying in anther vascular leads to the disorder of mechanism of glycelipid transforming in tapetal ceils, which triggers the tapetal cells degenerating ahead of time, and the latter gives rise to callus wall failing to degenerate in time. Therefore, the tetrads disintegrate and disappear ultimately due to lack of nutrition.

Comparative Studies on the Changes of Microtubule Distribution and Reorganization During the Meiotic Stages of Development in Normal (IR36) and a Temperature/photoperiod Sensitive Male Sterile Line (Peiai 64S) of Rice ( Oryza sativa )

Changes in the pattern of organization of microtubules in the meiotic stages of development of pollen (i.e. from pre-meiotic interphase to more or less metaphase I) of a normal (IR36) and a temperature/photoperiod sensitive male sterile line (Peiai 64S) of rice were studied using immunofluorescence confocal microscopy. In IR36, from pre-meiotic interphase to metaphase I, the pattern of microtubule distribution in the meiocytes underwent a series of changes. Some new organizational patterns of microtubules (that have not been described before) were observed during microsporogenesis, including the existence of a broad band of perinuclear microtubules at the diakinesis stage of development. The pattern of microtubule distribution in the meiocytes of the male sterile line, Peiai 64S, was quite different front that seen in IR36. In Peiai 64S, the microtubules showed abnormal patterns of distribution from pre-meiotic interphase to metaphase I. For example the broad band of perinuclear microtubules seen at diakinesis in IR36 was much disorganized and loosened in Peiai 64S. The spindles formed were also very abnormal and different from the normal spindle. The appearance of abnormal microtubule distribution in the early stages of microsporogenesis may contribute to the malformation and ultimate abortion of pollen in Peiai 64S.

Studies on Thermo-sensitivity of Ningxia Hot Pepper Male Sterile Line

[Objective] This study was conducted to identify the thermo-sensitivity of the male sterile line （NYB） of Ningxia hot pepper. [Method] The pol en abortion rates of pepper male sterile line under artificial y control ed temperature condition and nature temperature condition were measured and compared. [Result] The pol en abortion was the sign of NYB male sterility. The pol en abortion rate changed little under different temperature conditions, while pol en amount greatly reduced at low temperature, indicating that NYB was genetical y stable in field. [Conclusion] The re-sults can provide some theoretical references for the application of NYB pepper male sterile line in breeding work.

Molecular Markers and Agronomic Traits of a New Kind of Genic Male Sterile Material Mian 7AB-4-2 in Brassica napus L.

[ Objective] The study was to investigate the agronomic traits and breeding characteristics of genic male sterile material Mian 7AB-4- 2 in Brassica napus. [ Method] The differences in agronomic traits and polymorphisms in SSR markers, between the genic male sterile material Mian 7AB-4-2 in Brassica napus and its sisterly line Mian 7AB-4-1 were investigated by hybridization and molecular identification; and the percentage of sterile individuals of Mian 7AB-4-2 and of the hybrids with its sisterly line Mian 7AB-4-1 from test cross and back cross were also studled. [ Result] Mian 7AB-4-2 was not significantly different in agronomic traits from its sisterly line Mian 7AB-4-1 at 0.05 probability level. The per- centages of sterile individuals in the pollinated fertile Mian 7AB-4-2 plants were over 60%, and that in its sisterly line Mian 7AB-4-1 was about 25%. In test crosses with other nine sterile lines, Mian 7AB-4-1 kept the percentage of sterile individuals of sterile lines over 90%, and the percentage of sterile individuals from back cross over 80%. With regard to molecular markers, Mian 7AB-4-2 and its sisterly line Mian 7AB-4-1 were different in the band number from SSR primers a2 and E10. [ Conclusion] The results indicate that Mian 7AB-4-2 is helpful for rapeseed breeding, quickening the application of new materials in field breeding.

Study on Seed Vigor of Rice Photo-ThermoSensitive Genic Male Sterile(PTGMS) Line with Low Temperature Storage

To explore the change rule of seed vigor and seed application technology of rice PTGMS lines with low temperature storage,Zhun S,Y58S,Feng S and Guangzhan 63-4S stored in the seed bank with the temperature of 8-10 ℃ and with the relative humidity of 55% for different years were used as the materials to study the properties of seed vigor,growth and development in the field, etc. The re- sults showed that the germination potential, germination rate, germination index and vigor index of rice PTGMS lines seeds showed downward trends as the time of low temperature storage prolonged,and all of the germination rates of the seeds within five years were over 60%. The conductivity in the soaked solution of seeds was significantly increased with the prolonged low temperature storage time. Seedling rate,root length,white root number,base stem width and seedling dry weight of seeds showed downward trends as the time of low temperature storage prolonged. There was no significant difference between two integral seedling qualities of seeds within 4-6 years in low temperature storage. Compared with the germination rate of low temperature storage seeds soaking for 6,12 and 24 h,the appropriate soak time of long time low temperature storage or low vigor seeds was 12 h. A serious natural aging phenomenon occurred in low temperature storage seeds after out of the storage. The germination rate of seeds placed under natural condition for 50 d decreased obviously. The duration from seeding to heading of Zhun S,Y58S and Feng S shortened as the time of low temperature storage prolonged, while the vari- ation of panicle duration of all PTGMS lines was not obvious. In conclusion, rice PTGMS line seeds with low temperature storage in 5 years could still be used nor- mally. However.seeds after low temperature storage should be sowed as soon as possible, and the soaking time of low vigor seeds should be shortened,and the possibility that the decreased duration from seeding to heading should also be con- sidered.

Cytological Study on Zidao Type Cytoplasmic Male Sterile Line-Yingxiang A

Anthers of male sterile line and its maintainer line of Zidao type rice, Yingxiang A and Yingxiang B were studied by cytological methods, such as F-KI staining, silver nitrate staining and paraffin sections, It is found that in Yingxiang A, there are some abnormalities in meiosis, represented by unequal division at the meiotic phase; the abortion of CMS line arises at the meiotic phase and is almost completed at the phase of monomicrospore, but only a tiny amount of aborted mi- crospores are observed; the abortion is complete and thorough, and none normal functional pollens are found; the morphology of aborted pollens is similar to that of typically aborted pollens; the tapetal cells still exist in anthers from the microsporocyte stage to the tetrad stage, but they are abnormally hyperplastic. However, in Yingxiang B, there are no abnormalities in meiosis at the meiotic phase; the tapetal cells exist in anthers before the tetrad stage, but they disappear completely when the pollen are mature. It is indicated that the abortion of CMS Yingxiang line mainly occurs at the meiotic phase, and is basically completed at the phase of monomicrospore. The abortion is probably caused by the abnormally hyperplastic tapetal cells. The abnormalities in tapetal cells may affect the normal development of pollens, resulting in the emergence of abortion. In addition, the abortion in Yingxiang A is complete and thorough, which is of great significance in hybrid breeding.

Optimization of the Method to Obtain Effective Sterile Explants in Tissue Culture of Ardisia mamillata Hance

In the present study, we optimized the procedures to pretreat the parental material, to collect, sterilize and inoculate the explants in the tissue culture of Ar- disia mamillata Hance, so that more effective sterile explants can be obtained. The results revealed the optimal procedures. In detail, the parental materials were trans- ferred and pretreated in laboratory in February. The stem tips of new branches were collected in middle March, cleaned with eradicator and then rinsed with tap water for 1-2 h. After that, the explants were surface sterilized with 70% alcohol for 30 s, rinsed with sterile water 2-3 times, sterilized with 0.1% mercuric chloride for 4 min, rinsed with sterile water three times and sterilized with 0.1% mercuric chlo- ride for 4 min again. Finally, they were inoculated into medium after being rinsed with sterile water 5-8 times. By this method, the pollution rate of explants can be greatly reduced while their survival rate can be significantly improved.

Preliminary Discussion on Stigma Vitality of Sterile Line Rong 18A and Its Application in Seed Production

Rong 18A is an Indica three-line sterile line bred with M5B×D Xing B, and has the characteristics of stable sterility, high outcrossing rate, good combining ability and good quality. With Rong 18A as an experimental material and Ⅱ-32A, Jin 23A and Gang46 A as the CK materials, a seed setting rate estimation method by daily pollination was used for measuring its stigma vigor, to comparatively analyze the decreasing rates and lasting time of stigma vigor of various materials under natural condition. According to experimental results and combining with field breeding practice, the application of stigma vigor of sterile lines on hybrid rice seed production was discussed.