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Optimized protoplast isolation and transfection with a breakpoint:accelerating Cas9/sgRNA cleavage efficiency validation in monocot and dicot

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aBIOTECH 2024年第2期5卷 151-168页

作者： Debasmita Panda Subhasis Karmakar Manaswini Dash Swagat Kumar Tripathy Priya Das Sagar Banerjee Yiping Qi Sanghamitra Samantaray Pradipta Kumar Mohapatra Mirza J.Baig Kutubuddin A.Molla ICAR National Rice Research Institute CuttackOdisha 753006India Department of Botany Ravenshaw UniversityCuttackOdisha 753003India Department of plant Science and Landscape Architecture University of MarylandCollege ParkMD 20742USA

The CRISPR-Cas genome editing tools are revolutionizing agriculture and basic biology with their simplicity and precision ability to modify target genomic ***-predicted guide RNAs(gRNAs)often fail to induce efficient cleavage at target *** target loci are inaccessible due to complex chromatin ***,there is no suitable tool available to predict the architecture of genomic target sites and their ***,significant time and resources are spent on performing editing experiments with inefficient *** in vitro-cleavage assay could provide a rough assessment of gRNA efficiency,it largely excludes the interference of native genomic *** in-vivo testing gives a proper assessment of the cleavage ability of editing reagents in a native genomic ***,we developed a modified protocol that offers highly efficient protoplast isolation from rice,Arabidopsis,and chickpea,using a sucrose gradient,transfection using PEG(polyethylene glycol),and validation of single guide RNAs(sgRNAs)cleavage efficiency of *** have optimized various parameters for PEG-mediated protoplast transfection and achieved high transfection efficiency using our protocol in both monocots and *** introduced plasmid vectors containing Cas9 and sgRNAs targeting genes in rice,Arabidopsis,and chickpea *** dual sgRNAs,our CRISPRdeletion strategy offers straightforward detection of genome editing success by simple agarose gel *** sequencing of PCR products confirmed the editing efficiency of specific ***,we demonstrated that isolated protoplasts can be stored for up to 24/48 h with little loss of viability,allowing a pause between isolation and *** high-efficiency protocol for protoplast isolation and transfection enables rapid(less than 7 days)validation of sgRNA cleavage efficiency before proceeding with stable *** isolation and transfection method can als

关键词： Protoplast Genome editing Transgene-free edited plants Rice Chickpea

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Various Organic Nutrient Sources in Combinations with Inorganic Fertilizers Influence the Yield and Quality of Sweet Corn(Zea mays ***)in New Alluvial Soils of West Bengal,India

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Phyton-International Journal of Experimental Botany 2024年第4期93卷 763-776页

作者： Anindita Das Kanu Murmu Biplab Mitra Pintoo Bandopadhyay Ritesh Kundu Moupiya Roy Saleh Alfarraj Mohammad Javed Ansari Marian Brestic Akbar Hossain Department of Agronomy School of Agricultural SciencesJIS UniversityKalyaniNadiaWest Bengal741235India Department of Agronomy Bidhan Chandra Krishi ViswavidyalayaMohanpurWest Bengal741252India Department of Agronomy Uttar Banga Krishi ViswavidyalayaCoochbeharWest Bengal736101India Office of The Assistant director of Agriculture Gosaba BlockArampurSouth 24 ParganasWest Bengal743370India Department of Agricultural Statistics Bidhan Chandra Krishi ViswavidyalayaMohanpurWest Bengal741252India Zoology Department College of ScienceKing Saud UniversityRiyadh11451Saudi Arabia Department of Botany Hindu College Moradabad(Mahatma Jyotiba Phule Rohilkhand UniversityBareilly)MoradabadUttar Pradesh244001India Institute of plant and Environmental Sciences Slovak University of AgricultureTr.A.Hlinku 2Nitra94901Slovak Division of Soil Science Bangladesh Wheat and Maize Research InstituteDinajpur5200Bangladesh

Nutrient management plays a crucial role in the yield and quality of sweet corn.A field experiment was conducted in consecutive two kharif seasons in 2018 and 2019 to investigate the effect of various organic sources of nutrients in combination with inorganic sources on the yield and quality of sweet corn under new alluvial soils of West Bengal,*** were:T_(1):Control(without fertilizers);T_(2):100%recommended dose(RDF)of chemical fertilizers(CF)(RDF CF_(100%));T_(3):100%recommended dose of N(RDN)through vermicompost(VC)(RDN VC_(100%));T_(4):50 RDN through CF+50%RDN through VC(RDN CF_(50%)+RDN VC50%);T_(5):50%RDF through CF+50%RDN through organic source(OS)1,Soligro(Ascophyllum nodosum)granular(RDN CF_(50%)+RDN OS_(150%));T6:50%RDN through CF+50%RDN through OS 2,Bioenzyme(liquid)(RDN CF_(50%)+RDN OS250%);T7:50%RDN through CF+50%RDN through OS 3,Opteine(Ascophyllum nodosum)filtrate[RDN CF_(50%)+RDN OS350%];T8:50%RDN through VC+50%RDF through OS 1,Soligro(Ascophyllum nodosum)granular(RDN VC50%+RDN OS_(150%)).The OS of fertilizers were VC,SoliGro Gr(OS1)(Ascophyllum nodosum),Bioenzyme liquid(OS2),and Opteine(Ascophyllum nodosum)filtrate(OS3).The inorganic source was traditional CF applied at the RDF(150:75:75 kg ha^(−1) of N:P2O5:K2O).The VC was used to supply 100%RDN as one source or 50%RDN when combined with CF or *** fruit yield(10.75 and 10.79 t ha^(−1) in 2018 and 2019,respectively)was recorded when RDF was substituted through CF only,being statistically at par with 50%CF+50%VC on a nitrogen equivalent basis(9.92 and 10.00 t ha^(−1) in 2018 and 2019,respectively)and 100%VC(8.22 and 8.32 t ha^(−1) in 2018 and 2019,respectively).Compared to chemical sources of nutrients,VC-based treatments produced a larger percentage of large-size cob(>25 cm).The 100%VC increased antioxidant(8.35 and 8.45 mg g^(−1)),carotenoid(0.59 and 0.61 mg/100 g),and phenol(55.06 and 55.02 mg 100 g^(−1))content compared with its 50%dose in combination with other *** s

关键词： Biostimulants Ascophyllum nodosum crop quality crop yield sweet corn

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Spatial variation of carbon dioxide fluxes in corn and soybean fields

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Agricultural Sciences 2012年第8期3卷 986-995页

作者： Jerry L. Hatfield Timothy B. Parkin laboratory director and plant physiologist USDA-ARS National Laboratory for Agriculture and the Environment University Blvd Ames USA Soil Microbiologist USDA-ARS National Laboratory for Agriculture and the Environment University Blvd Ames USA

Spatial variation of soil carbon dioxide (CO2) flux during a growing season within corn and soybean canopies has not been quantified. These cropping systems are the most intense in the United States and the potential for carbon (C) sequestration in these systems through changes in soil management practices create an opportunity for reduction in greenhouse gas emissions;however, the need to understand the variation in fields is critical to evaluating changes in management systems. A study was designed to evaluate the spatial variation in soil CO2 fluxes along two transects in corn and soybean fields. Samples were collected every 5 m along a 100 m transect between the rows of the crop and also along a transect in which the plants had been removed to reduce the potential of root respiration. Soil CO2 fluxes were collected at each position with air temperature, soil temperature at 0.05 m, and soil water content (0 - 0.06 m). At the end of the season, soil samples for the upper 0.1 m were collected for soil organic C content, pH, sand, silt, and clay contents. On each day measurements were made, the observed CO2 emissions were scaled by dividing the CO2 flux at each position by the mean CO2 flux of the entire transect. Observed CO2 fluxes were signifycantly larger in the row than in the fallow position for both crops. There were no differences between the corn and soybean fallow transects;however, the corn row samples were larger than the soybean row samples. No consistent spatial patterns were observed in the CO2 fluxes or any of the soil properties over the course of the study. When the CO2 flux data were combined over the season, there was a significant spatial pattern in the fallow transects for both crops but not for the row transects. Sampling for CO2 flux values in cropping systems has to consider the presence of a crop canopy and the amount of root respiration.

关键词： Spatial Variation Transects Soil Organic Matter Soil Temperature Soil Moisture Soil Texture

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Conserved H3K27me3-associated chromatin looping mediates physical interactions of gene clusters in plants

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Journal of Integrative plant Biology 2023年第8期65卷 1966-1982页

作者： Linhua Sun Yuxin Cao Zhu Li Yi Liu Xiaochang Yin Xing Wang Deng Hang He Weiqiang Qian National Key laboratory of Wheat Improvement Peking University Institute of Advanced Agricultural SciencesShandong Laboratory of Advanced Agricultural Sciences in WeifangWeifang 261325China School of Advanced Agriculture Sciences Peking UniversityBeijing 100871China School of plant Science and Food Security Tel Aviv UniversityTel Aviv 6997801Israel

Higher-order chromatin organization is essential for transcriptional regulation,genome stability maintenance,and other genome *** evidence has revealed significant differences in 3D chromatin organization between plants and ***,the extent,pattern,and rules of chromatin organization in plants are still *** this study,we systematically identified and characterized long-range chromatin loops in the Arabidopsis 3D *** identified hundreds of long-range cis chromatin loops and found their anchor regions are closely associated with H3K27me3 epigenetic ***,we demonstrated that these chromatin loops are dependent on Polycomb group(PcG)proteins,suggesting that the Polycomb repressive complex2(PRC2)complex is essential for establishing and maintaining these novel *** most of these PcG-medicated chromatin loops are stable,many of these loops are tissue-specific or dynamically regulated by different ***,tandemly arrayed gene clusters and metabolic gene clusters are enriched in anchor ***-range H3K27me3-marked chromatin interactions are associated with the coregulation of specific gene ***,we also identified H3K27me3-associated chromatin loops associated with gene clusters in Oryza sativa and Glycine max,indicating that these long-range chromatin loops are conserved in *** results provide novel insights into genome evolution and transcriptional coregulation in plants.

关键词： Arabidopsis gene clusters Hi-C long-range chromatin loops H3K27me3

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Morphological and Genetical Variability among Rhizoctonia solani Isolates Causing Sheath Blight Disease of Rice

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Rice science 2016年第1期23卷 42-50页

作者： Zakiah Rahman MONI Md Ansar ALI Md Shahidul ALAM Md Asif RAHMAN Md Rejwan BHUIYAN Md Salim MIAN Khandakar Md IFTEKHARUDDAULA Md Abdul LATIF Mohammad Ashik Iqbal KHAN plant Breeding Division Bangladesh Rice Research Institute director Research Bangladesh Rice Research Institute Department of Botany Rajshahi University Department of Genetics and plant Breeding Bangabandhu Sheikh Mujibur Rahman Agricultural University plant Pathology Division Bangladesh Rice Research Institute

Eighteen isolates of Rhizoctonia solani collected from infected rice plants in four different locations of Bangladesh were studied by using morphological characters and molecular markers. Anastomosis study with a reference isolate confirmed that all the isolates belonged to R. solani. Significant variation was observed in sclerotial size, shape and distribution. Un-weighted pair group method with arithmetic mean dendrogram constructed based on the Gower＇s general similarity coefficient showed that these isolates were grouped into four clusters at the 0.68 similarity coefficent according to morphological characters. Cluster I was a major cluster consisting of 13 isolates, while clusters Ⅱ to Ⅳ consisted of 1 or 2 isolates. Analyses by variable number of tandem repeat and amplified fragment length polymorphism markers showed that the isolates were grouped into five and three clusters at a similarity coefficient of 0.64 and 0.69, respectively. Although most of the variability was found between isolates from different regions as expected, significant variation was observed within the isolates collected from similar agro-ecological regions. Our results suggest the presence of different races of R. solani within the same local geographic regions.

关键词： rice Rhizoctonia solani fungal variability molecular marker morphological character

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Two haplotype-resolved,gap-free genome assemblies for Actinidia latifolia and Actinidia chinensis shed light on the regulatory mechanisms of vitamin C and sucrose metabolism in kiwifruit

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Molecular plant 2023年第2期16卷 452-470页

作者： Xue Han Yilin Zhang Qiong Zhang Ni Ma Xiaoying Liu Wenjing Tao Zhiying Lou Caihong Zhong Xing Wang Deng Dawei Li Hang He Peking University Institute of Advanced Agricultural Sciences Shandong Laboratory of Advanced Agricultural Sciences in WeifangShandong 261325China School of Advanced Agricultural Sciences and School of Life Sciences Peking UniversityBeijing 100871China Key laboratory of plant Germplasm Enhancement and Specialty Agriculture Wuhan Botanical GardenThe Chinese Academy of SciencesWuhanHubei 430074China State Key laboratory of Cellular Stress Biology School of Life SciencesFaculty of Medicine and Life SciencesXiamen UniversityXiamen 361102China

Kiwifruit is a recently domesticated horticultural fruit crop with substantial economic and nutritional value,especially because of the high content of vitamin C in its *** this study,we de novo assembled two telomere-to-telomere kiwifruit genomes from Actinidia chinensis var.‘Donghong’(DH)and Actinidia latifolia‘Kuoye’(KY),with total lengths of 608327852 and 640561626 bp for 29 chromosomes,*** a burst of structural variants involving inversion,translocations,and duplications within 8.39 million years,the metabolite content of DH and KY exhibited differences in saccharides,lignans,and vitamins.A regulatory ERF098 transcription factor family has expanded in KY and Actinidia eriantha,both of which have ultra-high vitamin C *** each assembly phased into two complete haplotypes,we identified allelic variations between two sets of haplotypes,leading to protein sequence variations in 26494 and 27773 gene loci and allele-specific expression of 4687 and 12238 homozygous gene *** metabolome and transcriptome changes during DH fruit development revealed the same dynamic patterns in expression levels and metabolite contents;free fatty acids and flavonols accumulated in the early stages,but sugar substances and amino acids accumulated in the late *** AcSWEET9b gene that exhibits allelic dominance was further identified to positively correlate with high sucrose content in *** with wild varieties and other Actinidia species,AcSWEET9b promoters were selected in red-flesh kiwifruits that have increased fruit sucrose content,providing a possible explanation on why red-flesh kiwifruits are ***,these two gap-free kiwifruit genomes provide a valuable genetic resource for investigating domestication mechanisms and genome-based breeding of kiwifruit.

关键词： kiwifruit genome telomere-to-telomere allele-specific expression vitamin C sugar metabolism

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Haplotype Diversity at Sub1 Locus and Allelic Distribution Among Rice Varieties of Tide and Flood Prone Areas of South-East Asia

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Rice science 2017年第4期24卷 198-206页

作者： A.S.M.MASUDUZZAMAN Md.Maksudul HAQUE A.K.M.SHAMSUDDIN M.A.SALAM Md.Ansar ALI plant Breeding Division Bangladesh Rice Research InstituteGazipur 1701Bangladesh Scientific Officer(Golden Rice) Plant Breeding DivisionBangladesh Rice research InstituteGazipur 1701Bangladesh Department of Genetics and plant Breeding Bangladesh Agricultural UniversityMymensingh 2202Bangladesh director Research Bangladesh Rice Research InstituteGazipur 1701Bangladesh

Single nucleotide polymorphisms and restriction digestion-based haplotype variations among 160 flood prone rice varieties were analyzed with enzymes Alu I and Cac8 I to generate polymorphisms at Sub1A and Sub1C loci （conferring submergence tolerance）, respectively. Haplotype associated with phenotype was used to study the haplotype variations at Sub1A and Sub1C loci and to determine their functional influence on submergence tolerance and stem elongation. Three patterns at Sub1A locus, Sub1A0 （null allele）, Sub1A1 （does not cut） and Sub1A2 （one SNP）, and four patterns at Sub1C locus, Sub1C1, Sub1C2, Sub1C3 and Sub1C4, were generated. Both tolerant Sub1A1 and intolerant Sub1A2 had the same length, but the difference was presence of a restriction site in the Sub1A2, but absent at the Sub1A1. Further, two types of polymorphism were detected at the Sub1C, one included major length polymorphisms （165, 170 and 175 bp） and the other was a single restriction site at different position. Eight haplotypes （different combinations of the two loci）, A1C1, A1C2, A1C4, A2C2, A2C4, A0C2, A0C3 and A0C4, were detected among 160 varieties. Haplotype A1C1 was comparatively more related to haplotypes A1C2 and A1C4, having the same Sub1A allele, and these haplotypes were found only in Bangladeshi, Sri Lankan and Indian varieties. Most tolerant varieties in A1C1 haplotype showed slow elongation, having tolerant specific Sub1A1 and Sub1C1 alleles. Further, the varieties Madabaru and Kottamali （A2C2） also showed moderate level of tolerance without Sub1A1 allele. These varieties were different with FR13A and also suspected to carry different novel tolerant genes at other loci. These materials could be used for hybridization with Sub1 varieties for pyramiding additional tolerant specific alleles into a single genotype for improving submergence tolerance in rice.

关键词： haplotype Sub1 allele single nucleotide polymorphism submergence

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A scientific heritage in plant physiology from an older generation

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Science China(Life Sciences) 2012年第12期55卷 1125-1126页

作者： YUAN Ming State Key laboratory of plant Physiology and Biochemistry Department of Plant Sciences College of Biological Sciences China Agricultural University Beijing 100193 China

Scientific knowledge passes from generation to generation. The scientific achievements of one generation not only deepen our understanding of nature, but also

关键词：植物生理学科学遗产明尼苏达大学教育家科学知识科学成果现代科学生理学家

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plant biotic interactions

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Journal of Integrative plant Biology 2016年第4期58卷 282-283页

作者： Hailing Jin Special Issue EditorUC President's Chair director of Genetics Genomics and Bioinformatics Graduate ProgramCenter for Plant Cell BiologyInstitute for Integrative Genome BiologyUniversity of CaliforniaRiversideUSA

plants are constantly under attack by pathogens,pests,and parasites,resulting in severe consequences on global food production and human *** pathogens and pests find their ways to invade and communicate with their hos... 详细信息

关键词： communicate constantly sophisticated consequences microbial fight biotic evolved pathogen volatile

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Feeding and oviposition preference of Helopeltis theivora （Hemiptera： Miridae） on tea in Northeast India

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Insect Science 2006年第6期13卷 485-488页

作者： MANTU BHUYAN PRANAB R. BHATTACHARYYA Division of plant Sciences and Ecology Regional Research Laboratory Jorhat. Assam India

Feeding and oviposition preference studies of Helopeltis theivora Waterhouse, on leaves and stems of tea, Camellia sinensis L.（O）Kuntze, revealed that the insect mostly preferred the second leaf for feeding in comparison to the first, and the third leaf and stems; while the fifth instar nymphs were the most voracious feeders among the life stages, producing themost and largest feeding lesions. The insect preferred to oviposit mainly on stems and very few eggs are found on leaves.

关键词： feeding preference Helopeltis theivora lesion diameter oviposition tea tea mosquito bug

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