Morphological and cytological assessments reveal pollen degradation causes pollen abortion in cotton cytoplasmic male sterility lines

Background Understanding the mechanism of male sterility is crucial for producing hybrid seeds and developing sterile germplasm resources.However,only a few cytoplasmic male sterility(CMS)lines of cotton have been produced due to several challenges,like inadequate variation of agronomic traits,incomplete sterility,weak resilience of restorer lines,and difficulty in combining strong dominance.Therefore,the morphological and cytological identification of CMS in cotton will facilitate hybrid breeding.Results Two F_(2) segregating populations of cotton were constructed from cytoplasmic male sterile lines(HaA and 01A,maternal)and restorer lines(HaR and 26R,paternal).Genetic analysis of these populations revealed a segregation ratio of 3:1 for fertile to sterile plants.Phenotypic analysis indicated no significant differences in traits of flower bud development between sterile and fertile plants.However,sterile plants exhibited smaller floral organs,shortened filament lengths,and anther atrophy on the flowering day in comparison with the fertile plants.When performed scanning electron microscopy(SEM),the two F_(2) populations revealed morphological variations in the anther epidermis.Cellular analysis showed no significant differences in pollen development before pollen maturation.Interestingly,between the pollen maturation and flowering stages,the tapetum layer of sterile plants degenerated prematurely,resulting in abnormal pollen grains and gradual pollen degradation.Conclusion The results of this study suggest that fertility-restoring genes are controlled by a single dominant gene.Sterile plants exhibit distinctive floral morphology,which is characterized by stamen atrophy and abnormal anthers.Pollen abortion occurs between pollen maturity and flowering,indicating that premature tapetum degradation may be the primary cause of pollen abortion.Overall,our study provides a theoretical basis for utilizing CMS in hybrid breeding and in-depth investigation of the dominant configuration of cotton hybrid combinations,mechanisms of sterility,and the role of sterile and restorer genes.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Flowering Characteristics of Japonica Rice Male Sterile Lines Adaptive to High Temperature and Summer Drought

[Objective] This study was conducted to screen japonica rice male sterile lines with good flowering characteristics under high temperature and summer drought. [Method] The flowering habits of 23 japonica male sterile lines, such as flowering peak, single-panicle flowering duration, single-plant flowering duration, en- closed glume rate, glume-opening angle, the distance between glumes, stigma exer- tion rate and sigma vitality, were compared with II-32A as a control under above weather condition. [Result] The flowering characteristics had significant difference a- mong the tested rice lines. Compared with the control, three japonica rice male ster- ile lines 35478A, 35489A and 35502A had better flowering traits, such as earlier flowering peak, longer single-panicle flowering duration, longer single-plant flowering duration, lower rate of enclosed glumes, larger glume angle and larger distance be- tween glumes. 35478A performed its flowering peak from 10：00-11：00 am, 1.5 h earlier than the control; 35489A exhibited higher stigma exertion rate, up to 76.23%; and 35502A had higher stigma vitality, up to 84.68%, which kept longer time than that of the CK. [Conclusion] The results will provide high-quality resources for the breeding of new japonica rice varieties with high-yield and high-temperature toler- ance.

Recurrent Selection for Improving Quality Characters of Male Sterile Restorer Lines of Brassica napus

[Objective] This study aimed to improve the quality characters of the male sterile restorer lines of Brassica napus by recurrent selection. [Method] The dominant male sterile alleles were used as outcross medium to be transformed with the favourable genes from thirteen materials, and finally excellent restorer materials were bred by recurrent selection. After that, the genetic diversity, genetic gains, fatty acid chain length and unsaturarion ratios of the improved populations were analyzed. [Result] The genetic diversity and genetic gains were obviously improved by the recurrent selection. Nine materials whose oil content exceeding 50% and 14 materials whose protein content exceeding 30% were acquired, and crossed with sterile lines. The comparative experiment showed that there were 11 combinations whose oil content was obviously enhanced compared with the control. [Conclusion] Recurrent selection is an effective way to improve the quality characters of the objective pop- ulations and breed favourable male sterile restorer lines of B. napus.

Characterization of Reverse Thermo-sensitive Genic Male Sterile Lines in Cold Regions of China

Yannong S, a reverse thermo-sensitive genic male sterile （TGMS） line, is sterile at high temperature and fertile at low temperature. In the present study, the fertility of Yannong S and the japonica reverse TGMS lines derived from it was in- vestigated in Harbin, Heilongjiang from 2013 to 2015. The results showed that Yan- nong S was completely male sterile in July and August （the hottest months of the year in Harbin）, and the seed setting rate of bagged panicles was 0. We also in- ferred that the critical temperature for inducing the sterility of Yannong S was higher than 28 ℃. Most of the japonica reverse TGMS lines derived from Yannong S were completely aborted in July and August, with a seed setting rate of 0. It can be con- cluded that the reverse TGMS lines derived from Yannong S are genetically stable lines that have a long period of sterility in cold regions, so they may be the ideal genetic materials for the development and in-situ seed production of japonica hybrid rice.

Effect of High Temperature on Outcrossing Characteristics at the Fertility Sensitive Period of Photo-thermo Sensitive Genic Male Sterile (PTGMS) Rice Lines

Four sterile lines （Peiai64S, Y58S, Guangzhan 63-2S and H638S） and the restoring line R527 were used as materials. Five temperature gradients （24, 27, 30, 33 and 36 ℃ in artificial climate chamber） and the natural temperature （as control） were treated to the four sterile lines for 6 d in the fertility sensitive period of heading to flowering stage, respectively, to study the effects of temperature on physiological biochemical characteristics of young panicles and outcrossing characteristics. The results showed that the percentages of exerted stigma of Peiai 64S and Y58S were the highest at 27 ℃, which were 86.81% and 86.06%, respectively, while the percentages of exerted stigma of Guangzhan 63-2S and H638S were the highest at 24 ℃, which were 76.24% and 81.76%, respectively; the stigma viability of Peiai 64S, Y58S, Guangzhan 63-2S and H638S were the best at 24 ℃, which were 1.96, 2.12, 1.74 and 1.94, respectively; the outcrossing rates of Peiai 64S, Guangzhan 63-2S and H638S were the highest at 24 ℃, which were 58.87%, 54.22% and 50.50%, respectively, while the outcrossing rate of Y58S was the highest at 27 ℃, which was 58.96%; and the contents of peroxidase （POD） of the four sterile lines increased significantly at 33 ℃ compared with the control, while the contents of malondialdehyde （MDA） and proline increased significantly at 36 ℃ compared with the control. There were differences in temperature sensitivity between the male sterile lines, and the 24 ℃ treatment during the sensitive period was the best for the fertility sensitive period of Peiai 64S, while 27 ℃ was the best temperature for Y58S, Gangzhan 63-2S and H638S.

Preliminary Study on Inheritance of Stigma Exertion in Wheat Thermo-photo Sensitive Genic Male Sterile Line

Stigma exertion is one of the key factors for improving the outcrossing ability of wheat thermo-photo sensitive genic male sterile（TPSGMS） line. A DH population derived from K239S/K92 S was constructed to investigate the inheritance of stigma exertion. K239 S and K92 S are TPSGMS lines with higher and lower stigma exertion rates（SER）, respectively. The SERs of parents, reciprocal crosses and the DH population were evaluated for two consecutive years. The results showed that no significant difference was observed in SER between F1 s of K239S/K92 S and K92S/K239 S,implying that stigma exertion was a trait controlled by nuclear gene（s）. In the DH population, the segregation of low and high SERs fitted to a ratio of 3 ∶1 by Chisquare test, suggesting that the stigma exertion of K239 S was controlled by one pair of recessive genes. In addition, the effects of temperature and humidity on the expression of stigma exertion were also discussed.

Advances in DNA methylation and its role in cytoplasmic male sterility in higher plants

The impact of epigenetic modifications like DNA methylation on plant phenotypes has expanded the possibilities for crop development.DNA methylation plays a part in the regulation of both the chromatin structure and gene expression,and the enzyme involved,DNA methyltransferase,executes the methylation process within the plant genome.By regulating crucial biological pathways,epigenetic changes actively contribute to the creation of the phenotype.Therefore,epigenome editing may assist in overcoming some of the drawbacks of genome editing,which can have minor off-target consequences and merely facilitate the loss of a gene’s function.These drawbacks include gene knockout,which can have such off-target effects.This review provides examples of several molecular characteristics of DNA methylation,as well as some plant physiological processes that are impacted by these epigenetic changes in the plants.We also discuss how DNA alterations might be used to improve crops and meet the demands of sustainable and environmentally-friendly farming.

Creation of Male Sterile Line in Tobacco With HSP70 Anti-sense Fragment

In order to create the Male Sterile Line in tobacco, the anti-sense fragment of HSP70 gene was linked to anther specific expression promoter TA29 and the reconstructed vector was transformed into tobacco by Agrobacterium mediated transformation, and the transformants were then screened. Gus and spot blotting hybridization analysis of the transformants indicated that anti-sense fragment of HSF70 gene had been integrated into tobacco genome and expressed, thus the male sterile tobacco line was obtained. Microscope observation of anther and pollen showed that pistils of transgenic tobacco were normal, whereas anthers and pollens were fairly abortive in the same transgenic tobacco flower, comparing with pistils and stamens in control plants. The ratio of HSI：＇70 protein before and after heat shock in mitochondrial was found to be 1.39 in control tobacco plants and 1.01 in transgenic tobacco sterile lines. This is suggested that the anti-sense gene fragment of HSP70 can effectively inhibit the expression of HSP70 protein and lead to transgenic male sterility in tobacco flowers. The assay provided a new genetic engineering method for male sterility creation in plants.

Identification of a Cytoplasmic Male Sterile Line NEA in Brassica napus L.and Its Genetic Studies

Male sterile NEA plants were identified in progenies of the radiated 92P x Aggregate-silique in Brassica napus L. in 1993. Their progeny plants from test crossing and open pollination were 100% male sterile. The double-low male sterile lines JL-4 and JL-18 were bred through successive backcrossing of the double -low variety No.4 and No. 18 in Brassica napus L.to NEA. Restorer lines 6720 and 6722 with significant heterosis in F1 were developed. The results from investigating the restoring-ntaintaining relationship and inheritance of the restorer gene indicated that JL-4 and JL-18 were different from both PolCMS and Shan 2A type, and their restorer gene was controlled by a pair of dominant genie genes.

Generation of male-sterile soybean lines with the CRISPR/Cas9 system

Soybean [Glycine max(L.) Merr.] provides a rich source of plant protein and oil worldwide. The commercial use of transgenic technology in soybean has become a classical example of the application of biotechnology to crop improvement. Although genetically modified soybeans have achieved commercial success,hybrid soybean breeding is also a potential way to increase soybean yield. Soybean cytoplasmic malesterile(CMS) lines have been used in three-line hybrid breeding systems, but their application to exploiting soybean heterosis has been limited by rare germplasm resource of sterile lines. The generation of various genetic diversity male-sterile soybean lines will help to overcome the shortcoming. In this study,we used targeted editing of AMS homologs in soybean by CRISPR/Cas9 technology for the first time to generate stable male-sterile lines. Targeted editing of GmAMS1 resulted in a male-sterile phenotype,while editing of GmAMS2 failed to produce male-sterile lines. GmAMS1 functions not only in the formation of the pollen wall but also in the controlling the degradation of the soybean tapetum.CRISPR/Cas9 technology could be used to rapidly produce stable male-sterile lines, providing new sterile-line materials for soybean hybrid breeding systems.