Development of Insect-Resistant Hybrid Rice by Introgressing the Bt Gene from Bt Rice Huahui 1 into Ⅱ-32A/B,a Widely Used Cytogenic Male Sterile System

Huahui 1 is an elite transgenic male sterile restorer line of wild rice abortive-type that expresses a Bacillus thuringiensis （Bt） δ-endotoxin and provides effective and economic control oflepidopteran insects. To exploit Huahui 1 to develop a new Bt rice, the insertion site of the Bt gene was determined by thermal asymmetric interlaced PCR （TAIL-PCR）. Bt was located in the promoter region ofLOC. Os10g10360, approximately 5.35 Mb from the telomere of the short arm of chromosome 10. For the first time, a Bt cytoplasmic male sterile （CMS） system was developed by introgressing Bt from Huahui 1. The recipient CMS system used consisted of Indonesia paddy rice-type II-32B （maintainer line） and II-32A （male sterile line）. Marker-assisted selection was used to increase selection efficiency in the backcrossing program. In BC5F1, the Bt plant 85015-8 was selected for further analyses, as it had the highest SSR marker homozygosity. In addition, the linkage drag of the foreign Bt gene in 85015-8 was minimized to 8.01-11.46 Mb. The foreign Bt gene was then delivered from 85015-8 into II-32A. The resultant Bt II-32A and Bt II-32B lines were both resistant to lepidopteran in field trials, and agronomic traits were not disturbed. The maintainability of II-32B, and the male sterility and general combining ability of II-32A, were not affected by the Bt introgression. This study demonstrates a simple and fast approach to develop Bt hybrid rice.

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.

Establishment on Isolated Microspore Culture Optimized System for Restorer of New Cytoplasmic Male Sterile (NER) of Brassica napus L.

[Objective]The aim was to establish optimized system for NER isolated microspore culture of Brassica napus L.. [Method]Twenty varieties of NER of Brassica napus were grown under uncontrolled temperature and light conditions,and their isolated microspore from anthers were used as explants in vitro culture. The influencing factors of microspore culture were preliminarily studied. [Result]The difference of genotypes was important influencing factors to embryoid yield. The embryoid yield increased by supplementing with 6-BA and NAA,culturing in solid-liquid double layer medium with activated charcoal; The difference was not significant of embryoid yield between culturing in medium supplemented with colchicines and the CK. The rates of cotyledonous embryoids directly developed into normal plantlets increased through enriching with 0.1-0.2 mg/L NAA and being treated on slim illumination two days before being inducted into normal plantlets. [Conclusion]The technical system of microspore culture of restorer of new cytoplasmic male sterile （NER） was established,which and lays a foundation for accelerating genotype purification of NER introgressive line.

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.

DETECTION OF PRIMARY TISSUE FAILURE POTENTIAL OF BIOPROSTHETIC HEART VALVES IN A STERILE PULSE DUPLICATOR SYSTEM

This paper discusses results of anatomic analogy and transmission line model for designing a sterile pulse duplication system for bioprostheses. By simulating physiological flow and pressure pulses as well as the input impedance of the human arterial system Pulsatile function testing allows us to study the hydrodynamic performances and the leaflet action of each valve. This inspection enables us to identify and reject those valves exhibiting leaflet prolapse or lazy leaflet or others believed to contribute to Primary tissue failure. Consequently, the risk of valve complications is reduced

Spatiotemporal profiles of gene activity in stamen delineate nucleo-cytoplasmic interaction in a male-sterile somatic cybrid citrus

Cytoplasmic male sterility(CMS)has long been used to produce seedless fruits in perennial woody crops like citrus.A male-sterile somatic cybrid citrus(G1+HBP)was generated by protoplast fusion between a CMS callus parent‘Guoqing No.1’Satsuma mandarin(Citrus unshiu,G1)and a fertile mesophyll parent Hirado Buntan pummelo(Citrus grandis,HBP).To uncover the male-sterile mechanism of G1+HBP,we compared the transcriptome profiles of stamen organ and cell types at five stages between G1+HBP and HBP,including the initial stamen primordia,enlarged stamen primordia,pollen mother cells,tetrads,and microspores captured by laser microdissection.The stamen organ and cell types showed distinct gene expression profiles.A majority of genes involved in stamen development were differentially expressed,especially CgAP3.2,which was downregulated in enlarged stamen primordia and upregulated in tetrads of G1+HBP compared with HBP.Jasmonic acid-and auxin-related biological processes were enriched among the differentially expressed genes of stamen primordia,and the content of jasmonic acid biosynthesis metabolites was higher in flower buds and anthers of G1+HBP.In contrast,the content of auxin biosynthesis metabolites was lower in G1+HBP.The mitochondrial tricarboxylic acid cycle and oxidative phosphorylation processes were enriched among the differentially expressed genes in stamen primordia,meiocytes,and microspores,indicating the dysfunction of mitochondria in stamen organ and cell types of G1+HBP.Taken together,the results indicate that malfunction of mitochondria-nuclear interaction might cause disorder in stamen development,and thus lead to male sterility in the citrus cybrid.

Establishment of new convenient two-line system for hybrid production by targeting mutation of OPR3 in allopolyploid Brassica napus

The two-line pollination control system,which usually depends on the utilization of thermosensitive or photoperiod genic male-sterile lines,has been widely used in various crops.However,this system is susceptible to instability issues caused by uncontrollable weather fluctuations.A stable and handy two-line pollination control system is highly desirable in many crop species for heterosis exploitation.Oxophytodienoic acid reductase 3(OPR3)was proven to be involved in jasmonate biosynthesis.In the present study,CRISPR/Cas9(Clustered Regularly Interspaced Short Palindromic Repeat)was utilized to mutate two OPR3 homologs in Brassica napus.After two OPR3 homologs were simultaneously mutated,mutants exhibited complete male sterility,and fertility could be easily restored by exogenous MeJA treatment.Hybrids produced from crosses between the opr3 sterile lines and normal varieties exhibited heterosis.This new two-line system based on OPR3 mutation provides higher stability and convenience than traditional systems.By using exogenous MeJA treatment to restore fertility,the system enables more precise control of male fertility transition,which has great potential to significantly contribute to the maneuverable production of hybrid seeds in rapeseed as well as other Brassica species crops.

Improving plasma sterilization by constructing a plasma photocatalytic system with a needle array corona discharge and Au plasmonic nanocatalyst

Efficient sterilization by a plasma photocatalytic system(PPS)requires strong synergy between plasma and photocatalyst to inactivate microorganisms while suppressing the formation of secondary pollutants.Here,we report that a PPS constructed from a needle array corona discharge and Au/TiO2plasmonic nanocatalyst could remarkably improve the sterilization of Escherichia coli(E.coli)and alleviate formation of the discharge pollutant O3.At 6 kV,the combination of corona discharge and Au/TiO2achieves sterilization efficiency of 100%within an exposure time of 5 min.At 5 kV and an exposure time of 8 min,the presence of Au/TiO2improves sterilization efficiency of the corona discharge from 73%to 91%and reduces the O3concentration from 0.38 to 0.04 ppm,whereas the presence of TiO2reduces the sterilization efficiency and O3concentration to 66%and 0.17 ppm,respectively.The Au/TiO2in the PPS enables a uniform corona discharge,enhances the interaction between plasma,E.coli and nanocatalysts,and suppresses the formation of O3.Further,the Au/TiO2can be excited by ultraviolet-visible light emitted from the plasma to generate electron-hole pairs,and thus contributes to the formation of reactive radicals and the oxidative inactivation of E.coli.The PPS constructed from a needle array corona discharge and Au-based plasmonic nanocatalyst provides a promising approach for developing high-efficiency sterilization techniques.

Analysis of the Effect of Quality Control Circle on the Central Sterile Supply Department

Objective:To analyze the effect of quality control circle on the central sterile supply department(CSSD).Methods:The control group and the observation group each consisted of 180 instruments received by the sterilization supply center from January to March 2023 and 11 CSSD staff.The control group underwent routine management while quality control circle was implemented in the observation group.The quality of work,disinfection and sterilization qualification rates,disinfection and sterilization of various instruments,cleaning indicators,and management satisfaction of both groups were compared.Results:The observation group scored higher in terms of work quality,the qualification rate of disinfection and sterilization in each link,the disinfection and sterilization of instruments,and cleaning indicators compared to the control group.Besides,the management satisfaction of the observation group was higher than that of the control group(P<0.05).Conclusion:A quality control circle ensures the quality of work,improves the cleaning,disinfection,and sterilization of instruments of the CSSD,and improves the management satisfaction of the CSSD staff.

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.

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.

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.

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.

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.

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.

Transcriptional Regulation of 10 Mitochondrial Genes in Different Tissues of NCa CMS System in Brassica napus L. and Their Relationship with Sterility

Northern blot analysis was conducted with mitochondrial RNA from seedling leaves, floral buds, and developing seeds of NCa CMS, maintainer line and fertile F1 using ten mitochondrial genes as probes. The results revealed that 9 out of the 10 mitochondrial genes, except for atp6, showed no difference in different tissues of the corresponding materials of NCα CMS system and that they might be constitutively expressed genes. Eight genes, such as orf139, orf222, atpl, cox1, cox2, cob, rm5S, and rm26S, showed no difference among the three tissues of all the materials detected. So the expression of these eight genes was not regulated by nuclear genes and was not tissue-specific. The transcripts of atp9 were identical among different tissues, but diverse among different materials, indicating that transcription of atp9 was neither controlled by nuclear gene nor tissue-specific. Gene atp6 displayed similar transcripts with the same size among different tissues of all the materials but differed in abundance among tissues of corresponding materials and its expression might be tissue-specific under regulation of nuclear gene. Moreover, three transcripts of orf222 were detected in the floral buds of NCa cms and fertile F1, but no transcript was detected in floral buds of the maintainer line.The transcription of orf139 was similar to that of orf222 but only two transcripts of 0.8 kb and 0.6 kb were produced. The atp9 probe detected a single transcript of 0.6 kb in NCa cms and in maintainer line and an additional transcript of 1.2 kb in fertile F1. The relationship of expression of orf222, orf139, and atp9 with NCa sterility was 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.

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.

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.