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.

A novel Ag/ZnO core-shell structure for efficient sterilization synergizing antibiotics and subsequently removing residuals

The massive use of antibiotics has led to the aggravation of bacterial resistance and also brought environmental pollution problems.This poses a great threat to human health.If the dosage of antibiotics is reduced by increasing its bactericidal performance,the emergence of drug resistance is certainly delayed,so that there's not enough time for developing drug resistance during treatment.Therefore,we selected typical representative materials of metal Ag and semiconductor ZnO nano-bactericides to design and synthesize Ag/ZnO hollow core-shell structures(AZ for short).Antibiotics are grafted on the surface of AZ through rational modification to form a composite sterilization system.The research results show that the antibacterial efficiency of the composite system is significantly increased,from the sum(34.7%+22.8%-57.5%)of the antibacterial efficiency of AZ and gentamicin to 80.2%,net synergizes 22.7%,which fully reflects the effect of 1+1>2.Therefore,the dosage of antibiotics can be drastically reduced in this way,which makes both the possibility of bacterial resistance and medical expenses remarkably decrease.Subsequently,residual antibiotics can be degraded under simple illumination using AZ-self as a photocatalyst,which cuts off the path of environmental pollution.In short,such an innovative route has guiding significance for drug resistance.

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.

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.

Application of Molecular Markers Linking to Cytoplasmic Male Sterile Loci to Assist Maintainer Line Selection and Their Selection Efficiency in Welsh Onion (Allium fistulosum L.)

Cytoplasmic male sterility exists widely in most natural populations of welsh onion （Alliumfistulosum L.）, which makes it possible to breed out many male sterile lines for heterosis utilization. Unfortunately, the breeding of cytoplasmic male sterility in welsh onion has a little progress due to the limitation of its biological characteristic and traditional selection approach. To study the feasibility and the efficiency of utilizing marker assisted selection for male sterile lines in welsh onion, one SCAR marker, SCS13, and one RAPD marker, S2002400, which could distinguish between N and S cytoplasm in several welsh onion cultivars, were identified. The two markers were then confirmed by Southern blotting, and used to screen the N or S cytoplasm of individual plants in seven welsh onion cultivars in this study. Male sterile and fertile plants were evaluated by aceto-carmine dying. The frequency of N-cytoplasmic plants and maintainer genotype was calculated in the seven open populations of welsh onion. The minimum number of plants needed to identify a maintainer was evaluated to be 95% reliable. Results showed that 20 to 80% decrease of crosses and self-crosses for identifying a maintainer genotype could be achieved by the marker-assisted selection compared with traditional selection method. It was proved that the molecular markers could precisely identify cytoplasmic types individually, performed by one generation of cross and two generations of testcrosses and self-crosses. Finally, several maintainer genotype plants were selected with the help of the two markers in the seven cultivars. The screened markers could assist and accelerate sterile and maintainer lines selection with less labor and cost.

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.

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.

Synergistic roles of LAX1 and FZP in the development of rice sterile lemma

Rice florets are subtended by two sterile lemmas,whose origin and biological functions have not been studied extensively.Here we demonstrate that two putative transcription factors,LAX PANICLE1(LAX1)and FRIZZY PANICLE(FZP),synergistically control the development of sterile lemmas.Both LAX1 and FZP are previously known for their roles in panicle and floret development.Disruption of either LAX1 or FZP greatly reduces the number of floret development.We generated new lax1 mutants(lax1-c)using CRISPR/Cas9 gene editing technology.In addition to the expected lax panicle phenotypes,we noticed that a significant number of spikelets of lax1-c developed elongated sterile lemmas.Moreover,our characterization of lax1-RNAi plants also revealed sterile lemma phenotypes similar to lax1-c mutants.We isolated a weak allele of fzp(fzp-14)in a genetic screen for lax1–1 enhancers.The fzp-14 lax1–1 double mutants completely eliminated flower development.Interestingly,the isolated fzp-14 produced spikelets with elongated sterile lemmas.Furthermore,fzp-14 was haploid-insufficient in the lax1–1 background whereas fzp-14 heterozygous plants were indistinguishable from wild type plants.The lax1–1 fzp-14+/−also developed elongated sterile lemma as observed in lax1-c,lax1-RNAi,and fzp-14,suggesting that LAX1 and FZP synergistically control sterile lemma development.

Development and Application of SCAR Markers for Discriminating Cytoplasmic Male Sterile Lines from Their Cognate Maintainer Lines in Indica Rice

The DNAfragments about 1 600 bp were amplified using random amplified polymorphism DNA （RAPD） primer OPAl2 with the templates of mitochondrial DNA of Zhenshan 97A and Zhenshan 97B, and were sequenced. The nucleotide sequences and lengths of the fragments from Zhenshan 97A and Zhenshan 97B showed no difference. The precise length of the fragment was 1 588 bp. Sequence characterized amplification region （SCAR） primers were then developed to discriminate the cytoplasmic male sterile （CMS） lines and their maintainer lines. A specific 1 588 bp fragment could be amplified with SCAR primers, CHI19F2/CHI19R2 and CHI20F3/CHI23R3, in the mitochondrial DNA of Zhenshan 97A, but not Zhenshan 97B. Furthermore, the specific fragment could be also amplified from the total DNA from green leaf tissues of Zhenshan 97A with SCAR primers, but not Zhenshan 97B. With the corresponding primers, the specific fragment could also be amplified from the total DNA of green leaves of other two CMS lines with wild abortive type cytoplasm （CMS-WA）, namely Zhenpin A and Tianfeng A, but not in their maintainer lines. Moreover, using total DNA as template, each of the four pairs of SCAR primers could also be used to amplify the 1 588 bp fragment in CMS-ID （Indonesia paddy type） line 11-32A but not in 11-32B, and the specific fragment was amplified from the DNA of both F1 and F2 seedlings of Shanyou 63. The results of detecting the genetic purity of a man-made mixture of the seeds of Zhenshan 97A using CHI20F3/CHI23R3 were completely consistent with the phenotypes. Taken together, these results indicated that the specific 1 588 bp-fragment amplified by CHI20F3/CHI23R3 was the unique amplification products of CMS mitochondrial DNA, and could be used to distinguish CMS-WA and CMS-ID lines from their corresponding maintainer lines at the seedling stage.

Comparative Transcriptome Analysis between Cytoplasmic Male-sterile Line and Its Maintainer During the Floral Bud Development of Pepper

The male-sterile line has been largely used in the hybrid seed production of pepper, which can effectively improve the efficiency of hybrid seed production. However, the formation mechanism of male sterility in pepper remains unclear. In the present study, we compared the gene expression patterns between pepper cytoplasmic male sterile line 9704 A and its maintainer 9704 B during floral bud development using RNA sequencing technology. A total of 547 976 and 2 416 Differentially Expressed Genes(DEGs) were identified in the stage S1, S2 and S3, respectively,and more than 70% of the DEGs were down-regulated in the sterile line. Gene Ontology(GO), and Kyoto Encyclopedia of Genes and Genomes(KEGG) enrichment analyses were performed to further understand the functions of these identified DEGs. The results showed that the DEGs were mainly enriched in pathways of starch and sucrose metabolism, pentose and glucuronate interconversions. A number of genes, such as MS1, PME5, ATPB, and lots of transcription factors were found down-regulated in the sterile line, and we also identified a series of genes with large differences in expression patterns between sterile line and maintainer line. Collectively, our findings laid a foundation for further molecular breeding in pepper and provided new insights into its mechanism underlying the male sterility.

Comparisons on Genetic Diversity among the Isonuclear-Alloplasmic Male Sterile Lines and Their Maintainer Lines in Rice

Four sets of rice isonuclear-alloplasmic lines including 16 male sterile lines and their maintainer lines were analyzed by using 91 pairs of SSR primers to study the genetic diversity of nuclear genome and their relative relationships. A total of 169 alleles were detected in the 16 lines, with a frequency of polymorphic loci of 53.85% and an average number of alleles per locus of 1.8, and the average gene diversity was 0.228. Four sets of the isonuclear-alloplasmic male sterile lines shared 146 identical alleles, corresponding to 86.39% of the total alleles; meanwhile, there are 23 different alleles among the tested materials, being 13.61% of the total alleles. On average, 78.70% identical alleles and 21.30% different alleles of the total alleles were detected between the isonuclear-alloplasmic male sterile lines and their maintainer lines. There were 53.85% identical alleles and 46.15% different alleles of the total alleles among the homozygous allonucleus male sterile lines. The fingerprints were established for some male sterile lines and maintainer lines. All the materials tested were divided into three groups at the 0.2 genetic distance based on the cluster analysis. Eight lines of Huanong A and Huayu A （including Huanong B and Huayu B） were in the first group, four lines of Kezhen A （including Kezhen B） in the second group, and four lines of Zhenshan 97A （including Zhenshan 97B） in the third group. For the isonuclear-alloplasmic male sterile lines, the similarity coefficient between Y （Yegong） type and WA （wild abortive） type or between CW （Raoping wild rice） and WA type reached 87-98%.

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.

Effect of power supply parameters on discharge characteristics and sterilization efficiency of magnetically driven rotating gliding arc

Plasma sterilization is a new generation of high-tech sterilization method that is fast,safe,and pollution free.It is widely used in medical,food,and environmental protection fields.Home air sterilization is an emerging field of plasma application,which puts higher requirements on the miniaturization,operational stability,and operating cost of plasma device.In this study,a novel magnetically driven rotating gliding arc(MDRGA)discharge device was used to sterilize Lactobacillus fermentation.Compared with the traditional gas-driven gliding arc,this device has a simple structure and a more stable gliding arc.Simulation using COMSOL Multiphysics showed that adding permanent magnets can form a stable magnetic field,which is conducive to the formation of gliding arcs.Experiments on the discharge performance,ozone concentration,and sterilization effect were conducted using different power supply parameters.The results revealed that the MDRGA process can be divided into three stages:starting,gliding,and extinguishing.Appropriate voltage was the key factor for stable arc gliding,and both high and low voltages were not conducive to stable arc gliding and ozone production.In this experimental setup,the sterilization effect was the best at 6.6 kV.A high modulation duty ratio was beneficial for achieving stable arc gliding.However,when the duty ratio exceeded a certain value,the improvement in the sterilization effect was slow.Therefore,considering the sterilization effect and energy factors comprehensively,we chose 80%as the optimal modulation duty ratio for this experimental device.

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.

Fine mapping and cloning of the sterility gene Bra2Ms in nonheading Chinese cabbage(Brassica rapa ssp.chinensis)

The application of a male-sterile line is an ideal approach for hybrid seed production in non-heading Chinese cabbage(Brassica rapa ssp.chinensis).However,the molecular mechanisms underlying male sterility in B.rapa are still largely unclear.We previously obtained the natural male sterile line WS24-3 of non-heading Chinese cabbage and located the male sterile locus,Bra2Ms,on the A2 chromosome.Cytological observations revealed that the male sterility of WS24-3 resulted from disruption of the meiosis process during pollen formation.Fine mapping of Bra2Ms delimited the locus within a physical distance of about 129 kb on the A2 chromosome of B.rapa.The Bra039753 gene encodes a plant homeodomain(PHD)-finger protein and is considered a potential candidate gene for Bra2Ms.Bra039753 was significantly downregulated in sterile line WS24-3 compared to the fertile line at the meiotic anther stage.Sequence analysis of Bra039753 identified a 369 bp fragment insertion in the first exon in male sterile plants,which led to an amino acid insertion in the Bra039753 protein.In addition,the 369 bp fragment insertion was found to cosegregate with the male sterility trait.This study identified a novel locus related to male sterility in non-heading Chinese cabbage,and the molecular marker obtained in this study will be beneficial for the marker-assisted selection of excellent sterile lines in non-heading Chinese cabbage and other Brassica crops.

Ozone Sterilizer for Treatment and Health Care

Ozone is a green broad-spectrum bactericidal disinfectant, and a trace amount of ozone in the atmosphere makes many viruses and bacteria lose their biochemical activity and infectivity. Nature produces trace amounts of ozone in the air through lightning to purify the ecological environment. The product of ozone decomposition is oxygen, without secondary pollution. Ozone sterilizer is widely used in the epidemic prevention and control of intensive breeding farms and achieved remarkable results. If the concentration and action time of ozone can be accurately controlled, then ozone can quickly eliminate pathogens, without harming the normal cells in the human body. How to use medical ozone for epidemic prevention, treatment and health care is a subject worthy of serious study, which should arouse the attention of the experts in the field.

Differential RNA Editing of Mitochondrial Genes in WA-Cytoplasmic Based Male Sterile Line Pusa 6A, and Its Maintainer and Restorer Lines

RNA editing changes the nucleotides at the transcript level of mitochondrial genes which results in synthesis of functional proteins.This study was designed to find the editing sites which could be implicated in male fertility restoration and to develop editing based markers for differentiation of cytoplasmic male sterility and maintainer lines from each other.DNA and RNA from young panicles were isolated from three-line system of hybrid rice PRH10,wild abortive(WA)cytoplasm based male sterile(A line Pusa 6A),maintainer(B line Pusa 6B)and restorer(R line PRR78)lines.Pusa 6A and PRR78 having the same WA cytoplasm are allo-nuclear and iso-cytpolasmic lines.The genomic and cDNA amplicons for eight mitochondrial genes(18SrRNA,atp6,atp9,cobII,coxI,coxIII,nadI and rps3)were sequenced and compared.Differences in genomic and cDNA sequences were considered as editing.Two hundred and thirty editing sites having base substitution or insertion/deletion were identified with the highest in 18SrRNA(5.74%)and the lowest in coxI(0.60%).The highest editing sites were observed in fertile maintainer Pusa 6B followed by PRR78 and Pusa 6A,of which random five editing sites in five different rice mitochondrial transcripts namely atp9,cobII,coxIII,rps3 and 18SrRNA were chosen and validated through cleaved amplified polymorphism sequence(CAPS)analysis and found to be partially edited in four genes.The identical editing sites of different mitochondrial genes from maintainer and restorer lines might reflect their possible contribution to fertility restoration of sterile WA cytoplasm.

Studies of the Energy Release of Mitochondria from Sporophyte Cytoplasmic Male Sterile Rice by Differential Scanning Calorimetry

The differential scanning calorimetric (DSC)curves of the mitochondria isolated from two varieties of sporophyte cytoplasmic male sterile and their fertile lines of Yie Bai and Ma Xie type rice have been determined. The curves show that the energy is released continuously as temperature rise to 70℃. Some thermodynamic and kinetic parameters of the energy release of the mitochondria have been obtained. The presented results showed that the mitochondria from cytoplasmic male sterile rice released more heat and they had higher energy barrier, less rate, and more complicated mechanism than that of their fertile lines in the energy release process.

Breeding of Maintainer Line and Sterile Line Brown Planthopper Resistant Rice Using Marker-assisted Selection

Guangxi common wild rice variety brown planthopper highly resistant introgression line HS204 was taken as antigen donor material,hybridization,backcrossing,and molecular marker-assisted selection methods were adopted,to select maintainer line and sterile line materials,so as to provide excellent material foundation for resistance breeding of hybrid rice. Through the marker-assisted selection,it obtained 4 pieces of homozygous resistance gene maintainer line intermediate materials( 100B,101B,102B and 103B),and 2 pieces of resistant sterile line materials( 100A and 103A). All 10 combinations that have testcross with highly resistant sterile 100A showed higher level of brown planthopper resistance: 100A/R2586,100A/KR838,and 100A/KR527 had high resistance level,the others had low to intermediate resistance;100A/KR527,100A/R2586,100A/Minghui 63,100A/Fuhui 838 and 100A/Gui 99 combinations had yield per plant significantly higher than the control group( Teyou 7118),increasing by 14. 45%-49. 26%. The obtained resistant lines are expected to provide a better gene source for the breeding of resistant sterile lines of hybrid rice and the obtained resistant sterile lines can be directly used in the selection of three-line hybrid rice.