Pub Date : 2020-01-01DOI: 10.35248/2329-9029.20.8.246
D. Liang, P. Shuai, X. Xia, W. Yin
DNA methylation is an important epigenetic modification that has been implicated in many biological processes. Methylation changes during stress responses and developmental processes have been well studied. Here, we present a novel comparison of methylation patterns in two poplar species. We detected the global methylation patterns in leaves of Populus trichocarpa and Populus euphratica by methylated DNA immune precipitation sequencing (MeDIPseq). A total of eleven significant modules were detected in the two species. Our results indicate that the methylation of CG/CHH/CHG contexts was much more significant in P. trichocarpa than in P. euphratica, but methylation changes in response to saline stress were consistent in both species.
DNA甲基化是一种重要的表观遗传修饰，与许多生物过程有关。甲基化在应激反应和发育过程中的变化已经得到了很好的研究。在这里，我们提出了一个新的比较甲基化模式在两种杨树。利用甲基化DNA免疫沉淀测序技术(methylated DNA immune precipitation sequencing, MeDIPseq)检测了毛杨(Populus trichocarpa)和胡杨(Populus euphratica)叶片的甲基化模式。在两个物种中共检测到11个重要模块。我们的研究结果表明，毛藻中CG/CHH/CHG环境的甲基化要比胡杨显著得多，但在盐水胁迫下甲基化的变化在两种物种中是一致的。
{"title":"Genome-Wide Analysis of DNA Cytosine Methylation in Two Poplars","authors":"D. Liang, P. Shuai, X. Xia, W. Yin","doi":"10.35248/2329-9029.20.8.246","DOIUrl":"https://doi.org/10.35248/2329-9029.20.8.246","url":null,"abstract":"DNA methylation is an important epigenetic modification that has been implicated in many biological processes. Methylation changes during stress responses and developmental processes have been well studied. Here, we present a novel comparison of methylation patterns in two poplar species. We detected the global methylation patterns in leaves of Populus trichocarpa and Populus euphratica by methylated DNA immune precipitation sequencing (MeDIPseq). A total of eleven significant modules were detected in the two species. Our results indicate that the methylation of CG/CHH/CHG contexts was much more significant in P. trichocarpa than in P. euphratica, but methylation changes in response to saline stress were consistent in both species.","PeriodicalId":16778,"journal":{"name":"Journal of Plant Biochemistry & Physiology","volume":"94 1","pages":"1-6"},"PeriodicalIF":0.0,"publicationDate":"2020-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"90377779","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2019-02-01DOI: 10.4172/2329-9029.1000229
Dagnachew Bekele, K. Tesfaye, A. Fikre
Virus induced gene silencing (VIGS) is an effective technology that exploits an antiviral defense mechanism in plants. It is a recently developed gene transcript suppression technique for characterizing the function of plant genes. VIGS is rapid, efficient and specific system for transient gene silencing. The major steps in VIGS includes; engineering viral genomes to the appropriate viral vector to incorporate fragments of host genes that are targeted to be silenced, infecting the appropriate plant hosts and silencing the target genes as part of the defense mechanism of the plant against virus infection. The VIGS vector is a recombinant virus engineered to be able to carry a piece an endogenous gene from the host. During infection with the modified vector, the host’s defense reaction will be induced against the cloned host gene; a loss of function phenotype makes it possible to identify the function of the gene. The recombinant virus is introduced into plant cells through Agrobacterium tumefaciens mediated transient expression or in vitro transcribed RNA inoculation or direct DNA inoculation. The Trans gene is amplified along with the viral RNA by RNA dependent RNA polymerase generating dsRNA molecules. dsRNA is the triggering molecule of Post transcriptional gene silencing. VIGS as a reverse genetics tool for functional genomics studies presenting several advantages. Despite its great potential, many limitations remain to be overcome. In this review, the molecular mechanism in VIGS technology, its advanced application in plant functional genomics studies and the major limitation and potential future prospects were briefly discussed.
{"title":"Applications of Virus Induced Gene Silencing (VIGS) in Plant Functional Genomics Studies","authors":"Dagnachew Bekele, K. Tesfaye, A. Fikre","doi":"10.4172/2329-9029.1000229","DOIUrl":"https://doi.org/10.4172/2329-9029.1000229","url":null,"abstract":"Virus induced gene silencing (VIGS) is an effective technology that exploits an antiviral defense mechanism in plants. It is a recently developed gene transcript suppression technique for characterizing the function of plant genes. VIGS is rapid, efficient and specific system for transient gene silencing. The major steps in VIGS includes; engineering viral genomes to the appropriate viral vector to incorporate fragments of host genes that are targeted to be silenced, infecting the appropriate plant hosts and silencing the target genes as part of the defense mechanism of the plant against virus infection. The VIGS vector is a recombinant virus engineered to be able to carry a piece an endogenous gene from the host. During infection with the modified vector, the host’s defense reaction will be induced against the cloned host gene; a loss of function phenotype makes it possible to identify the function of the gene. The recombinant virus is introduced into plant cells through Agrobacterium tumefaciens mediated transient expression or in vitro transcribed RNA inoculation or direct DNA inoculation. The Trans gene is amplified along with the viral RNA by RNA dependent RNA polymerase generating dsRNA molecules. dsRNA is the triggering molecule of Post transcriptional gene silencing. VIGS as a reverse genetics tool for functional genomics studies presenting several advantages. Despite its great potential, many limitations remain to be overcome. In this review, the molecular mechanism in VIGS technology, its advanced application in plant functional genomics studies and the major limitation and potential future prospects were briefly discussed.","PeriodicalId":16778,"journal":{"name":"Journal of Plant Biochemistry & Physiology","volume":"27 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2019-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"90468901","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2019-01-01DOI: 10.35248/2329-9029.19.7.244
AboShama Hm, Atwa Mm
Double haploid (DH) plants Production is a valuable tool in plant breeding programs. Since conventional breeding takes long time for the development of newly improved cultivars, this technique reduces the time needed for this purpose. Practically, application of (DH) technology in potato breeding through androgenesis in Santana cultivar is possible by considering number of factors that influence androgenesis. Culture medium has to be considered in respect of constituents to fulfill the needs of culture target where N6 medium significantly showed better results (14.61%) if compared to MS medium (8.78%) anthers produced embryos. Auxin/cytokinin represented in this study by 2,4-D/BA showed better results in induction of embryogenesis when combined together at (2.0 mg/l 2,4-D and 0.5 mg/l BA) which resulted in (35.67%) anthers produced embryos rather than each one of them alone. Enhanced results were obtained after floral buds pretreatment with thermal chock with three different degrees (4°C, 25°C, 30°C) for two different exposure time (48 h and 72 h). N6 medium showed the highest percentage of anthers produced embryos (44%) when supplemented with 2 mg/l 2,4-D+0.5 mg/l BA, while pretreatment with 4°C for 72 h favored the embryos production (16.67%) followed by (14.00%) and (12.00%) at 25°C and 4°C for 48 h respectively with no significant difference among them. Pretreatment with 32°C for (48 h and 72 h) resulted in (4.67%) and (5.33%) respectively differed significantly form the low temperature. Silver nitrate has shown to be potent at 2 mg/l in inhibiting ethylene, increasing embryogenesis (60.0 %) and reducing the non-responsive anthers (6.67 %).
{"title":"Anther Culture in Potato (Solanum tuberosum L.) in vitro","authors":"AboShama Hm, Atwa Mm","doi":"10.35248/2329-9029.19.7.244","DOIUrl":"https://doi.org/10.35248/2329-9029.19.7.244","url":null,"abstract":"Double haploid (DH) plants Production is a valuable tool in plant breeding programs. Since conventional breeding takes long time for the development of newly improved cultivars, this technique reduces the time needed for this purpose. Practically, application of (DH) technology in potato breeding through androgenesis in Santana cultivar is possible by considering number of factors that influence androgenesis. Culture medium has to be considered in respect of constituents to fulfill the needs of culture target where N6 medium significantly showed better results (14.61%) if compared to MS medium (8.78%) anthers produced embryos. Auxin/cytokinin represented in this study by 2,4-D/BA showed better results in induction of embryogenesis when combined together at (2.0 mg/l 2,4-D and 0.5 mg/l BA) which resulted in (35.67%) anthers produced embryos rather than each one of them alone. Enhanced results were obtained after floral buds pretreatment with thermal chock with three different degrees (4°C, 25°C, 30°C) for two different exposure time (48 h and 72 h). N6 medium showed the highest percentage of anthers produced embryos (44%) when supplemented with 2 mg/l 2,4-D+0.5 mg/l BA, while pretreatment with 4°C for 72 h favored the embryos production (16.67%) followed by (14.00%) and (12.00%) at 25°C and 4°C for 48 h respectively with no significant difference among them. Pretreatment with 32°C for (48 h and 72 h) resulted in (4.67%) and (5.33%) respectively differed significantly form the low temperature. Silver nitrate has shown to be potent at 2 mg/l in inhibiting ethylene, increasing embryogenesis (60.0 %) and reducing the non-responsive anthers (6.67 %).","PeriodicalId":16778,"journal":{"name":"Journal of Plant Biochemistry & Physiology","volume":"30 1","pages":"1-10"},"PeriodicalIF":0.0,"publicationDate":"2019-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"79241647","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2019-01-01DOI: 10.35248/2329-9029.19.7.243
AboShama Hm, Atwa Mm
Recently, tissue culture based in vitro selection have been studied as cost-effective feasible tool for developing stress-tolerant plants. Somaclonal variation created in an experiment conducted with two potatoes (Solanum tuberosum L.) cultivars Santana and Spunta to create somaclonal variation by repetitious subcultures on MS medium for the purpose of evaluating this somaclonal variation for biotic stress resistance, the two cultivars were inoculated in vitro with four concentrates 1 × 102, 1 × 104, 1 × 106 and 1 × 108 cfu/ml Pectobacterium atrosepticum. Both cultivars showed significant reduction of disease severity index (DSI) at highest inoculum level 1 × 108 cfu/ml between pre and post subculture. Santana cv. DSI was 51.17% and 61.5% improved significantly to 25.17% and 44.17% for calcium treated and non-treated respectively, while in Spunta cv. DSI was 54.00% and 62.33% improved significantly to 32.67% and 46.17% for calcium treated and non-treated respectively. The highest DSI recorded in pre-subculture non-treated with Ca were 33.23% and 35.16% for Santana and Spunta cvs. respectively while the lowest DSI recorded in post subculture treated with Ca were 11.97% and 15.20% for Santana and Spunta cvs. respectively. In vivo inoculation of in vitro survived plantlets with the same inoculum levels showed better performance of Santana cv. over Spunta cv. at 1 × 104 ,1 × 106 cfu/ml with (20.33%, 33.33% ) and (34.00%, 42.67% ) respectively. Calcium nitrate improved plants tolerance by reducing DSI significantly from 66.33% to 35.00% at 1 × 108 cfu/ml for non-treated and treated respectively.
{"title":"In vitro Evaluation of Somaclonal Variation of Two Potato Cultivars Santana and Spunta for Resistance against Bacterial Blackleg Pectobacterium atrosepticum","authors":"AboShama Hm, Atwa Mm","doi":"10.35248/2329-9029.19.7.243","DOIUrl":"https://doi.org/10.35248/2329-9029.19.7.243","url":null,"abstract":"Recently, tissue culture based in vitro selection have been studied as cost-effective feasible tool for developing stress-tolerant plants. Somaclonal variation created in an experiment conducted with two potatoes (Solanum tuberosum L.) cultivars Santana and Spunta to create somaclonal variation by repetitious subcultures on MS medium for the purpose of evaluating this somaclonal variation for biotic stress resistance, the two cultivars were inoculated in vitro with four concentrates 1 × 102, 1 × 104, 1 × 106 and 1 × 108 cfu/ml Pectobacterium atrosepticum. Both cultivars showed significant reduction of disease severity index (DSI) at highest inoculum level 1 × 108 cfu/ml between pre and post subculture. Santana cv. DSI was 51.17% and 61.5% improved significantly to 25.17% and 44.17% for calcium treated and non-treated respectively, while in Spunta cv. DSI was 54.00% and 62.33% improved significantly to 32.67% and 46.17% for calcium treated and non-treated respectively. The highest DSI recorded in pre-subculture non-treated with Ca were 33.23% and 35.16% for Santana and Spunta cvs. respectively while the lowest DSI recorded in post subculture treated with Ca were 11.97% and 15.20% for Santana and Spunta cvs. respectively. In vivo inoculation of in vitro survived plantlets with the same inoculum levels showed better performance of Santana cv. over Spunta cv. at 1 × 104 ,1 × 106 cfu/ml with (20.33%, 33.33% ) and (34.00%, 42.67% ) respectively. Calcium nitrate improved plants tolerance by reducing DSI significantly from 66.33% to 35.00% at 1 × 108 cfu/ml for non-treated and treated respectively.","PeriodicalId":16778,"journal":{"name":"Journal of Plant Biochemistry & Physiology","volume":"19 1","pages":"1-10"},"PeriodicalIF":0.0,"publicationDate":"2019-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"84426896","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2019-01-01DOI: 10.35248/2329-9029.19.7.241
R. Al-Hazmi
The present investigation was implemented to search for high efficient bacterial strains in bio-remediating the toxic influence of Lead chloride (Pb Cl2) on faba bean plants grown in sandy soil supplemented with various concentrations of Pb Cl2. Five strains were isolated from Al-Taif, Al-Baha and Al-Madinah Al-Munawrah governorates from the rhizosphere of cucumber, fig and tomato plants respectively, KSA. Regarding efficiency of these isolated strains in remediating PbCl2 in the nutrient broth medium, the absorbed quantities ranged from 189 to 417 mg/l while the whole amount recorded in control was 612 mg/l. The isolated bacterial strains were genetically identified as Bacillus amyloliquefaciens subsp. plantarum Trigo Core 1448, Bacillus amyloliquefaciens subsp. plantarum UCMB 5033, Bacillus subtilis strain CCM 1999, Serratia marcescens strain WW4 and Bacillus amyloliquefaciens CC178. Bacillus amyloliquefaciens subsp. plantarum UCMB 5033 revealed the highest efficiency in absorbing Pb that reached 417 mg/l against 612 mg/l for the controls. Bioremediation of Pb-polluted soil with Bacillus amyloliquefaciens significantly increased faba bean plant height, plant fresh weight, dry weight, plant N, P and K concentrations compared to the non-inoculated treatments.
{"title":"Isolation and Identification of Five Lead Bio-Remediating Bacterial Strains","authors":"R. Al-Hazmi","doi":"10.35248/2329-9029.19.7.241","DOIUrl":"https://doi.org/10.35248/2329-9029.19.7.241","url":null,"abstract":"The present investigation was implemented to search for high efficient bacterial strains in bio-remediating the toxic influence of Lead chloride (Pb Cl2) on faba bean plants grown in sandy soil supplemented with various concentrations of Pb Cl2. Five strains were isolated from Al-Taif, Al-Baha and Al-Madinah Al-Munawrah governorates from the rhizosphere of cucumber, fig and tomato plants respectively, KSA. Regarding efficiency of these isolated strains in remediating PbCl2 in the nutrient broth medium, the absorbed quantities ranged from 189 to 417 mg/l while the whole amount recorded in control was 612 mg/l. The isolated bacterial strains were genetically identified as Bacillus amyloliquefaciens subsp. plantarum Trigo Core 1448, Bacillus amyloliquefaciens subsp. plantarum UCMB 5033, Bacillus subtilis strain CCM 1999, Serratia marcescens strain WW4 and Bacillus amyloliquefaciens CC178. Bacillus amyloliquefaciens subsp. plantarum UCMB 5033 revealed the highest efficiency in absorbing Pb that reached 417 mg/l against 612 mg/l for the controls. Bioremediation of Pb-polluted soil with Bacillus amyloliquefaciens significantly increased faba bean plant height, plant fresh weight, dry weight, plant N, P and K concentrations compared to the non-inoculated treatments.","PeriodicalId":16778,"journal":{"name":"Journal of Plant Biochemistry & Physiology","volume":"118 2 1","pages":"1-5"},"PeriodicalIF":0.0,"publicationDate":"2019-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"86460807","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2019-01-01DOI: 10.4172/2329-9029.1000232
G. Daniel, S. Krishnakumari
In recent years, there is an increasing interest in the study of free radicals since free radicals are the reason for various human diseases. Different forms of radicals are generated by human body as a result of certain metabolic pathways. Normally there is a balance in the levels of free radicals and antioxidants for proper physiological conditions. Overproduction of the free radicals causes oxidative damage to the biomolecules like lipids, DNA and proteins. This leads to chronic diseases like cardiovascular diseases, neuronal disease, cataract, cancer etc. The present study was designed to evaluate the potential of aqueous leaf extract of Eugenia uniflora as an antioxidant lead by using various in vitro models like lipid peroxidation (FTC and TBA method, total antioxidant capacity and reducing power scavenging assays using standard procedures. IC50 values were calculated respectively. In all these studies, a significant correlation existed between concentrations of the extract and percentage inhibition of free radicals. These results clearly indicated that leaf extract of Eugenia unflora could be a potential source of natural antioxidant and effective against free radical mediated diseases.
{"title":"Free Radical Scavenging Activity of Aqueous (Hot) Extract of Eugenia uniflora (L.) Leaves","authors":"G. Daniel, S. Krishnakumari","doi":"10.4172/2329-9029.1000232","DOIUrl":"https://doi.org/10.4172/2329-9029.1000232","url":null,"abstract":"In recent years, there is an increasing interest in the study of free radicals since free radicals are the reason for various human diseases. Different forms of radicals are generated by human body as a result of certain metabolic pathways. Normally there is a balance in the levels of free radicals and antioxidants for proper physiological conditions. Overproduction of the free radicals causes oxidative damage to the biomolecules like lipids, DNA and proteins. This leads to chronic diseases like cardiovascular diseases, neuronal disease, cataract, cancer etc. The present study was designed to evaluate the potential of aqueous leaf extract of Eugenia uniflora as an antioxidant lead by using various in vitro models like lipid peroxidation (FTC and TBA method, total antioxidant capacity and reducing power scavenging assays using standard procedures. IC50 values were calculated respectively. In all these studies, a significant correlation existed between concentrations of the extract and percentage inhibition of free radicals. These results clearly indicated that leaf extract of Eugenia unflora could be a potential source of natural antioxidant and effective against free radical mediated diseases.","PeriodicalId":16778,"journal":{"name":"Journal of Plant Biochemistry & Physiology","volume":"29 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2019-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"90422779","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2019-01-01DOI: 10.4172/2329-9029.1000233
Purity G. Limbua, M. Ngugi, Richard O. Oduor
A reproducible regeneration protocol for ICGV 12991, CG 7 and Red Valencia groundnut genotypes using Cotyledonary Node explants has been optimized. The effect of different BAP concentrations combined with either 2,4-D or TDZ was tested to determine optimum conditions for high shoot induction. Different BAP concentrations were tested to determine an optimum concentration for shoot elongation. Different NAA concentrations were similarly evaluated to determine the best concentration for rooting. Media containing combination of 5 mg/L BAP and 1 mg/L TDZ was the best concentration for shoot induction while media containing BAP at 5 mg/L was the best for elongation of shoots. NAA concentration of 1 mg/L gave the highest number of plants with roots. This works provides a very good protocol which will be beneficial during groundnut tissue culture as well as genetic transformation of groundnuts.
{"title":"In Vitro Regeneration Protocol of Kenyan Adapted Groundnut (Arachis hypogaea L.) Genotypes using Cotyledonary Node Explants","authors":"Purity G. Limbua, M. Ngugi, Richard O. Oduor","doi":"10.4172/2329-9029.1000233","DOIUrl":"https://doi.org/10.4172/2329-9029.1000233","url":null,"abstract":"A reproducible regeneration protocol for ICGV 12991, CG 7 and Red Valencia groundnut genotypes using Cotyledonary Node explants has been optimized. The effect of different BAP concentrations combined with either 2,4-D or TDZ was tested to determine optimum conditions for high shoot induction. Different BAP concentrations were tested to determine an optimum concentration for shoot elongation. Different NAA concentrations were similarly evaluated to determine the best concentration for rooting. Media containing combination of 5 mg/L BAP and 1 mg/L TDZ was the best concentration for shoot induction while media containing BAP at 5 mg/L was the best for elongation of shoots. NAA concentration of 1 mg/L gave the highest number of plants with roots. This works provides a very good protocol which will be beneficial during groundnut tissue culture as well as genetic transformation of groundnuts.","PeriodicalId":16778,"journal":{"name":"Journal of Plant Biochemistry & Physiology","volume":"16 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2019-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"73219696","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2019-01-01DOI: 10.4172/2329-9029.1000230
Ankita Shrestha, Ahamed Khan, N. Dey
The single RNA‐guided DNA recognition CRISPR-Cas9 method is a simple and powerful tool for targeted genome engineering. Here, we report the designing and testing of efficient caulimoviral promoter-derived binary vectors for performing genome editing employing the CRISPR-Cas9-gRNA system. For such targeted mutagenesis, we created binary transformation vectors to drive the expression of Cas9 by an efficient caulimoviral promoter, ‘M24’ isolated and characterized from the Mirabilis Mosaic Virus (MMV). The 20-nucleotide CRISPR guide (g)-RNAs were designed to induce targeted mutations in the CYP82E4-nicotine N-demethylase (nnd) gene of tobacco (Nicotiana tabacum). For editing the nnd gene, we employed a pair of gRNAs followed by the protospacer adjacent motif (PAM) targeting the first exon of the nnd-ORF. We evaluated the percent “indels” using tobacco protoplast cells where mutagenesis frequencies were recorded as 45% and 30% for the two targets respectively. A mutagenesis efficiency of 37% was obtained upon the simultaneous transfection of the two gRNAs in tobacco protoplasts. Successful demonstration of our caulimoviral-based CRISPR-Cas9-gRNA system bodes well for its near-term use as a potential and facile means to performing targeted genome editing in plants.
{"title":"CRISPR-Cas9-Mediated Editing of the CYP82E4-Nicotine N-Demethylase (nnd) Gene in Tobacco Protoplasts","authors":"Ankita Shrestha, Ahamed Khan, N. Dey","doi":"10.4172/2329-9029.1000230","DOIUrl":"https://doi.org/10.4172/2329-9029.1000230","url":null,"abstract":"The single RNA‐guided DNA recognition CRISPR-Cas9 method is a simple and powerful tool for targeted genome engineering. Here, we report the designing and testing of efficient caulimoviral promoter-derived binary vectors for performing genome editing employing the CRISPR-Cas9-gRNA system. For such targeted mutagenesis, we created binary transformation vectors to drive the expression of Cas9 by an efficient caulimoviral promoter, ‘M24’ isolated and characterized from the Mirabilis Mosaic Virus (MMV). The 20-nucleotide CRISPR guide (g)-RNAs were designed to induce targeted mutations in the CYP82E4-nicotine N-demethylase (nnd) gene of tobacco (Nicotiana tabacum). For editing the nnd gene, we employed a pair of gRNAs followed by the protospacer adjacent motif (PAM) targeting the first exon of the nnd-ORF. We evaluated the percent “indels” using tobacco protoplast cells where mutagenesis frequencies were recorded as 45% and 30% for the two targets respectively. A mutagenesis efficiency of 37% was obtained upon the simultaneous transfection of the two gRNAs in tobacco protoplasts. Successful demonstration of our caulimoviral-based CRISPR-Cas9-gRNA system bodes well for its near-term use as a potential and facile means to performing targeted genome editing in plants.","PeriodicalId":16778,"journal":{"name":"Journal of Plant Biochemistry & Physiology","volume":"81 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2019-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"85792285","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2019-01-01DOI: 10.35248/2329-9029.19.7.238
Narayani Shukla, Y. Varma
Superoxide dismutases (SODs) are ubiquitous metalloenzymes that constitute the first line of defense against reactive oxygen species. The role of SOD in neutralizing free radical production has already been documented. In the present investigation we have attempt to study kinetic properties of SODs in three drought sensitive and drought resistant varieties of H. vulgare. The results demonstrated that SOD displayed decreased affinities of substrate binding to their corresponding enzymes under drought conditions. The SOD isozymes profile was also analyzed and it was found that under drought conditions the only two SOD isozymes expressed as against three isozymes expressed under control. These data suggest that the decreased substrate binding affinity to SODs as well as decrease in one isozyme band of SOD under drought condition may be responsible for resistance. These findings may be used to develop drought resistance varieties of other crops.
{"title":"Enzymatic Analysis of Superoxide Dismutase (SOD) from Hordeum vulgare: Its Role in Drought Stress Tolerance","authors":"Narayani Shukla, Y. Varma","doi":"10.35248/2329-9029.19.7.238","DOIUrl":"https://doi.org/10.35248/2329-9029.19.7.238","url":null,"abstract":"Superoxide dismutases (SODs) are ubiquitous metalloenzymes that constitute the first line of defense against reactive oxygen species. The role of SOD in neutralizing free radical production has already been documented. In the present investigation we have attempt to study kinetic properties of SODs in three drought sensitive and drought resistant varieties of H. vulgare. The results demonstrated that SOD displayed decreased affinities of substrate binding to their corresponding enzymes under drought conditions. The SOD isozymes profile was also analyzed and it was found that under drought conditions the only two SOD isozymes expressed as against three isozymes expressed under control. These data suggest that the decreased substrate binding affinity to SODs as well as decrease in one isozyme band of SOD under drought condition may be responsible for resistance. These findings may be used to develop drought resistance varieties of other crops.","PeriodicalId":16778,"journal":{"name":"Journal of Plant Biochemistry & Physiology","volume":"39 1","pages":"1-4"},"PeriodicalIF":0.0,"publicationDate":"2019-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"87326879","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2019-01-01DOI: 10.4172/2329-9029.1000228
A. Al-Daoude, E. Al-shehadah, A. Shoaib, M. Jawhar, Arabi Mie
Spot blotch (SB), caused by the necrotrophic fungal pathogen Cochliobolus sativus, is an important disease of barley globally. Following transcriptional changes of salicylic acid (SA)-interacting/binding proteins during C. sativus infection may greatly advance understanding the defense crucial signaling pathways. In this study, changes of four known categories of defense; phosphorylation, ROS, PR proteins and nucleotide-binding sites encoded by genes involved in SA–mediated defense signaling networks were studied in compatible/incompatible barley-SB interactions. The functional categories showed significant differential accumulations when compared to the noninoculated controls, and they were primarily upregulated during fungal infection in the resistant cultivar compared with the susceptible one. However, SA profiling of resistant and susceptible cultivars indicated a reduction in its levels 72 hours post inoculation; therefore, we hypothesized that this signaling pathways may facilitate SB resistance. Furthermore, the expression of selected categories was induced earlier in resistant barley plants as in susceptible ones, supporting the hypothesis that a delayed defense response may occur in the C. sativus susceptible interaction.
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