Ratna Karan | University of Florida (original) (raw)
Papers by Ratna Karan
Gcb Bioenergy, May 23, 2020
This is an open access article under the terms of the Creative Commons Attribution License, which... more This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.
Biotechnology Journal, Mar 24, 2021
Precision genome editing by homology directed repair has tremendous potential for crop improvemen... more Precision genome editing by homology directed repair has tremendous potential for crop improvement. This study describes in planta homologous recombination mediated by CRISPR/Cas9 induced DNA double strand break in proximity to a single short (∼30 nt) homology arm. The efficiency of CRISPR/Cas9‐mediated recombination between two loxP sites was compared with Cre (Cyclization recombination enzyme) and codon‐optimized Cre‐mediated site‐specific recombination in sugarcane. A transgenic locus was generated with a selectable nptII coding sequence with terminator between two loxP sites located downstream of a constitutive promoter and acting as transcription block for the downstream promoter‐less gusA coding sequence with terminator. Recombination between the two loxP sites resulted in deletion of the transcription block and restored gus activity. This transgenic locus provided an efficient screen for identification of recombination events in sugarcane callus following biolistic delivery of Cre, codon‐optimized Cre, or the combination of sgRNA and Cas9 targeting the 5′ loxP site. The Cre codon optimized for sugarcane displayed the highest efficiency in mediating the recombination that restored gus activity followed by cre and CRISPR/Cas9. Remarkably the short region of homology of the loxP site cleaved by Cas9 (30 nt)‐mediated error‐free recombination in all 21 events from three different experiments that were analyzed by Sanger sequencing consistent with homology directed repair. These findings will inform rational design of strategies for precision genome editing in plants.
Plant Cell Tissue and Organ Culture, May 26, 2016
Sugarcane is a prime crop for commercial production of bioethanol and table sugar. Genetic engine... more Sugarcane is a prime crop for commercial production of bioethanol and table sugar. Genetic engineering is complementing traditional breeding in sugarcane improvement. Costs and delays associated with regulatory approval for a transgenic event are substantial impediments to the commercialization of transgenic crops. Generation of intragenic sugarcane events carrying additional sequences from sexually compatible sorghum resembles traditional plant breeding and may facilitate regulatory approval. Acetolactate synthase (ALS), is a key enzyme in the biosynthetic pathway of branched-chain amino acids, and also a target for ALS-inhibiting herbicides including sulfonylureas and imidazolinones. In this study, we developed an intragenic minimal expression cassette encoding a mutant sorghum acetolactate synthase gene (mALS) under transcriptional control of the sorghum ubiquitin promoter and sorghum HSP 3 0 UTR. mALS was delivered into tissue cultures of sugarcane cultivar CP 88-1762 by biolistic gene transfer. A suitable selection protocol for recovery of intragenic events was developed for the herbicide bispyribac sodium. Intragenic sugarcane events were identified by PCR, and qRT-PCR analysis revealed events with different levels of mALS expression. Expression of the mALS gene in sugarcane resulted in chlorsulfuron resistance, as demonstrated by lower injury level and increased plant height following application of chlorsulfuron compared to the control cultivar. The results demonstrated that the mALS gene from sorghum supports the production of intragenic, herbicide resistant sugarcane. This is the first report of an intragenic selectable marker for sugarcane.
Plant Molecular Biology, Mar 27, 2019
Background/objective Transgenic sugarcane technology will complement breeding in the development ... more Background/objective Transgenic sugarcane technology will complement breeding in the development of advanced cultivars and will benefit the global sugar and biofuel industries. In this study, we generated a highly expressed single copy locus with sequences for site specific recombination as a landing pad for transgene stacking.
Achieving sustainable cultivation of sugarcane Volume 2, 2018
Plant Cell, Tissue and Organ Culture (PCTOC), 2016
Sugarcane is a prime crop for commercial production of bioethanol and table sugar. Genetic engine... more Sugarcane is a prime crop for commercial production of bioethanol and table sugar. Genetic engineering is complementing traditional breeding in sugarcane improvement. Costs and delays associated with regulatory approval for a transgenic event are substantial impediments to the commercialization of transgenic crops. Generation of intragenic sugarcane events carrying additional sequences from sexually compatible sorghum resembles traditional plant breeding and may facilitate regulatory approval. Acetolactate synthase (ALS), is a key enzyme in the biosynthetic pathway of branched-chain amino acids, and also a target for ALS-inhibiting herbicides including sulfonylureas and imidazolinones. In this study, we developed an intragenic minimal expression cassette encoding a mutant sorghum acetolactate synthase gene (mALS) under transcriptional control of the sorghum ubiquitin promoter and sorghum HSP 3 0 UTR. mALS was delivered into tissue cultures of sugarcane cultivar CP 88-1762 by biolistic gene transfer. A suitable selection protocol for recovery of intragenic events was developed for the herbicide bispyribac sodium. Intragenic sugarcane events were identified by PCR, and qRT-PCR analysis revealed events with different levels of mALS expression. Expression of the mALS gene in sugarcane resulted in chlorsulfuron resistance, as demonstrated by lower injury level and increased plant height following application of chlorsulfuron compared to the control cultivar. The results demonstrated that the mALS gene from sorghum supports the production of intragenic, herbicide resistant sugarcane. This is the first report of an intragenic selectable marker for sugarcane.
Crop improvement is essentially required to provide food security and better nutrition globally f... more Crop improvement is essentially required to provide food security and better nutrition globally for human beings. Plant breeders are utilizing both conventional and modern breeding techniques to increase crop production and quality. Though in the genome-sequencing era, marker-assisted selection-based breeding reduced timespan required for developing new plant variety from ~ 25 to ~ 7 years. But plant breeders are still looking for appropriate molecular method to quickly and precisely improve specific traits in plants. Recently discovered genome editing methods have revolutionized available technology for crop improvements. Genome editing is a tool of genetic engineering, which using engineered nucleases edits DNA of an organism. In editing, DNA is inserted, deleted, or replaced in the genome using engineered or designer nucleases. These nucleases create site-specific double-strand breaks (DSBs) at precise locations in the genome. The induced DSBs are repaired through more frequent n...
Biotechnology Journal, 2021
Precision genome editing by homology directed repair has tremendous potential for crop improvemen... more Precision genome editing by homology directed repair has tremendous potential for crop improvement. This study describes in planta homologous recombination mediated by CRISPR/Cas9 induced DNA double strand break in proximity to a single short (∼30 nt) homology arm. The efficiency of CRISPR/Cas9‐mediated recombination between two loxP sites was compared with Cre (Cyclization recombination enzyme) and codon‐optimized Cre‐mediated site‐specific recombination in sugarcane. A transgenic locus was generated with a selectable nptII coding sequence with terminator between two loxP sites located downstream of a constitutive promoter and acting as transcription block for the downstream promoter‐less gusA coding sequence with terminator. Recombination between the two loxP sites resulted in deletion of the transcription block and restored gus activity. This transgenic locus provided an efficient screen for identification of recombination events in sugarcane callus following biolistic delivery of Cre, codon‐optimized Cre, or the combination of sgRNA and Cas9 targeting the 5′ loxP site. The Cre codon optimized for sugarcane displayed the highest efficiency in mediating the recombination that restored gus activity followed by cre and CRISPR/Cas9. Remarkably the short region of homology of the loxP site cleaved by Cas9 (30 nt)‐mediated error‐free recombination in all 21 events from three different experiments that were analyzed by Sanger sequencing consistent with homology directed repair. These findings will inform rational design of strategies for precision genome editing in plants.
Frontiers in Genome Editing, 2021
Sugarcane is the source of 80% of the sugar and 26% of the bioethanol produced globally. However,... more Sugarcane is the source of 80% of the sugar and 26% of the bioethanol produced globally. However, its complex, highly polyploid genome (2n = 100 – 120) impedes crop improvement. Here, we report efficient and reproducible gene targeting (GT) in sugarcane, enabling precise co-editing of multiple alleles via template-mediated and homology-directed repair (HDR) of DNA double strand breaks induced by the programmable nuclease CRISPR/Cas9. The evaluation of 146 independently transformed plants from five independent experiments revealed a targeted nucleotide replacement that resulted in both targeted amino acid substitutions W574L and S653I in the acetolactate synthase (ALS) in 11 lines in addition to single, targeted amino acid substitutions W574L or S653I in 25 or 18 lines, respectively. Co-editing of up to three ALS copies/alleles that confer herbicide tolerance was confirmed by Sanger sequencing of cloned long polymerase chain reaction (PCR) amplicons. This work will enable crop improv...
PLoS ONE, 2012
Background: Salinity is a major environmental factor limiting productivity of crop plants includi... more Background: Salinity is a major environmental factor limiting productivity of crop plants including rice in which wide range of natural variability exists. Although recent evidences implicate epigenetic mechanisms for modulating the gene expression in plants under environmental stresses, epigenetic changes and their functional consequences under salinity stress in rice are underexplored. DNA methylation is one of the epigenetic mechanisms regulating gene expression in plant's responses to environmental stresses. Better understanding of epigenetic regulation of plant growth and response to environmental stresses may create novel heritable variation for crop improvement. Methodology/Principal Findings: Methylation sensitive amplification polymorphism (MSAP) technique was used to assess the effect of salt stress on extent and patterns of DNA methylation in four genotypes of rice differing in the degree of salinity tolerance. Overall, the amount of DNA methylation was more in shoot compared to root and the contribution of fully methylated loci was always more than hemi-methylated loci. Sequencing of ten randomly selected MSAP fragments indicated gene-body specific DNA methylation of retrotransposons, stress responsive genes, and chromatin modification genes, distributed on different rice chromosomes. Bisulphite sequencing and quantitative RT-PCR analysis of selected MSAP loci showed that cytosine methylation changes under salinity as well as gene expression varied with genotypes and tissue types irrespective of the level of salinity tolerance of rice genotypes. Conclusions/Significance: The gene body methylation may have an important role in regulating gene expression in organ and genotype specific manner under salinity stress. Association between salt tolerance and methylation changes observed in some cases suggested that many methylation changes are not ''directed''. The natural genetic variation for salt tolerance observed in rice germplasm may be independent of the extent and pattern of DNA methylation which may have been induced by abiotic stress followed by accumulation through the natural selection process.
Journal of Heredity, 2013
Crop Science, 2012
Seed dormancy (SD) is a key domestication trait closely related to preharvest sprouting tolerance... more Seed dormancy (SD) is a key domestication trait closely related to preharvest sprouting tolerance. Wild and weedy rices (Oryza spp.) exhibit higher degrees of seed dormancy compared to the cultivated rice. Red rice (Oryza sativa L.), a major weed in the rice growing areas of the southern United States, was used to elucidate the genetic architecture of SD. Quantitative trait loci (QTL) analysis conducted in two recombinant inbred line (RIL) populations developed from the crosses involving two rice cultivars (Bengal and Cypress) and a red rice accession (PSRR-1) revealed six to seven QTL for seed dormancy, which accounted for 49 to 52% of the total phenotypic variance. The magnitude of the QTL contribution to phenotypic variance was infl uenced by genetic backgrounds. The majority of QTL had minor effects, except the QTL linked to Rc and Sdr4. The genetic architecture for seed dormancy in U.S. red rice was distinct compared with the earlier reported weedy accessions. Four QTL were mapped onto similar positions in both populations. Both cultivars and red rice contributed alleles for increased SD. Most of the digenic epistatic interactions involved loci other than the QTL with main effects. The nucleotide polymorphisms at the Sdr4 locus could not explain the phenotypic variation for seed dormancy in our materials. The variation in SD among the rice cultivars could be attributed to segregation of minor QTL, which may be exploited to improve preharvest sprouting tolerance.
BMC Plant Biology, 2012
Background Metallothioneins (MT) are low molecular weight, cysteine rich metal binding proteins, ... more Background Metallothioneins (MT) are low molecular weight, cysteine rich metal binding proteins, found across genera and species, but their function(s) in abiotic stress tolerance are not well documented. Results We have characterized a rice MT gene, OsMT1e-P, isolated from a subtractive library generated from a stressed salinity tolerant rice genotype, Pokkali. Bioinformatics analysis of the rice genome sequence revealed that this gene belongs to a multigenic family, which consists of 13 genes with 15 protein products. OsMT1e-P is located on chromosome XI, away from the majority of other type I genes that are clustered on chromosome XII. Various members of this MT gene cluster showed a tight co-regulation pattern under several abiotic stresses. Sequence analysis revealed the presence of conserved cysteine residues in OsMT1e-P protein. Salinity stress was found to regulate the transcript abundance of OsMT1e-P in a developmental and organ specific manner. Using transgenic approach, w...
BMC Plant Biology, 2012
Background: SUMO (Small Ubiquitin related Modifier) conjugation is a post translational regulator... more Background: SUMO (Small Ubiquitin related Modifier) conjugation is a post translational regulatory process found in all eukaryotes, mediated by SUMO activating enzyme, SUMO conjugating enzyme, and SUMO ligase for the attachment of SUMO to its target protein. Although the mechanism for regulation of SUMO conjugation pathway genes under abiotic stress has been studied to certain extent, the role of SUMO conjugating enzyme in improving abiotic stress tolerance to plant is largely unexplored. Here, we have characterized a SUMO conjugating enzyme gene 'SaSce9' from a halophytic grass Spartina alterniflora and investigated its role in imparting abiotic stress tolerance. Results: SaSce9 gene encodes for a polypeptide of 162 amino acids with a molecular weight of~18 kD and isoelectric point 8.43. Amino acid sequence comparisons of SaSce9 with its orthologs from other plant species showed high degree (~85-93%) of structural conservation among each other. Complementation analysis using yeast SCE mutant, Ubc9, revealed functional conservation of SaSce9 between yeast and S. alterniflora. SaSce9 transcript was inducible by salinity, drought, cold, and exogenously supplied ABA both in leaves and roots of S. alterniflora. Constitutive overexpression of SaSce9 in Arabidopsis through Agrobacterium mediated transformation improved salinity and drought tolerance of Arabidopsis. SaSce9 overexpressing Arabidopsis plants retained more chlorophyll and proline both under salinity and drought stress. SaSce9 transgenic plants accumulated lower levels of reactive oxygen under salinity stress. Expression analysis of stress responsive genes in SaSce9 Arabidopsis plants revealed the increased expression of antioxidant genes, AtSOD and AtCAT, ion antiporter genes, AtNHX1 and AtSOS1, a gene involved in proline biosynthesis, AtP5CS, and a gene involved in ABA dependent signaling pathway, AtRD22. Conclusions: These results highlight the prospect of improving abiotic stress tolerance in plants through genetic engineering of the sumoylation pathway. The study provides evidence that the overexpression of SaSce9 in plant can improve salinity and drought stress tolerance by protecting the plant through scavenging of ROS, accumulation of an osmolyte, proline, and expression of stress responsive genes. In addition, this study demonstrates the potential of the halophyte grass S. alterniflora as a reservoir of abiotic stress related genes for crop improvement.
GCB Bioenergy
This is an open access article under the terms of the Creative Commons Attribution License, which... more This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.
![Research paper thumbnail of {"__content__"=>"Transgene Pyramiding of Salt Responsive Protein 3-1 () and From L. Enhances Salt Tolerance in Rice.", "i"=>[{"__content__"=>"SaSRP3-1"}, {"__content__"=>"SaVHAc1"}, {"__content__"=>"Spartina alterniflora"}]}](https://attachments.academia-assets.com/72917306/thumbnails/1.jpg)
](Frontiers in plant science, 2018
The transgenic technology using a single gene has been widely used for crop improvement. But the ... more The transgenic technology using a single gene has been widely used for crop improvement. But the transgenic pyramiding of multiple genes, a promising alternative especially for enhancing complexly inherited abiotic stress tolerance, has received little attention. Here, we developed and evaluated transgenic rice lines with a single Salt Responsive Protein 3-1 () gene as well as pyramids with two-genes and Vacuolar H-ATPase subunit c1 () derived from a halophyte grass L. for salt tolerance at seedling, vegetative, and reproductive stages. The overexpression of this novel gene resulted in significantly better growth of with the recombinant plasmid under 600 mM NaCl stress condition compared with the control. During early seedling and vegetative stages, the single gene and pyramided transgenic rice plants showed enhanced tolerance to salt stress with minimal wilting and drying symptoms, improved shoot and root growth, and significantly higher chlorophyll content, relative water content,...
Scientific reports, Feb 1, 2018
Although flowering in rice has been extensively investigated, few studies focused on genetic inte... more Although flowering in rice has been extensively investigated, few studies focused on genetic interactions. Flowering evaluation of two recombinant inbred line (RIL) populations involving photo-insensitive rice cultivars, Bengal and Cypress, and a weedy rice accession, PSRR-1, under natural long-day (LD) conditions, revealed six to ten quantitative trait loci (QTLs) and a major QTL interaction. In addition to the validation of several previously cloned genes using an introgression lines (IL) population of PSRR-1, a few novel QTLs were also discovered. Analysis of the marker profiles of the advanced backcross lines revealed that Hd1 allele of PSRR-1 was responsible for the photoperiodic response in the near-isogenic lines (NILs) developed in both cultivar backgrounds. Based on the phenotypic and genotypic data of the NILs, and NIL mapping population and the transcript abundance of key flowering pathway genes, we conclude that Hd1 and its interaction with a novel gene other than Ghd7 p...
Gcb Bioenergy, May 23, 2020
This is an open access article under the terms of the Creative Commons Attribution License, which... more This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.
Biotechnology Journal, Mar 24, 2021
Precision genome editing by homology directed repair has tremendous potential for crop improvemen... more Precision genome editing by homology directed repair has tremendous potential for crop improvement. This study describes in planta homologous recombination mediated by CRISPR/Cas9 induced DNA double strand break in proximity to a single short (∼30 nt) homology arm. The efficiency of CRISPR/Cas9‐mediated recombination between two loxP sites was compared with Cre (Cyclization recombination enzyme) and codon‐optimized Cre‐mediated site‐specific recombination in sugarcane. A transgenic locus was generated with a selectable nptII coding sequence with terminator between two loxP sites located downstream of a constitutive promoter and acting as transcription block for the downstream promoter‐less gusA coding sequence with terminator. Recombination between the two loxP sites resulted in deletion of the transcription block and restored gus activity. This transgenic locus provided an efficient screen for identification of recombination events in sugarcane callus following biolistic delivery of Cre, codon‐optimized Cre, or the combination of sgRNA and Cas9 targeting the 5′ loxP site. The Cre codon optimized for sugarcane displayed the highest efficiency in mediating the recombination that restored gus activity followed by cre and CRISPR/Cas9. Remarkably the short region of homology of the loxP site cleaved by Cas9 (30 nt)‐mediated error‐free recombination in all 21 events from three different experiments that were analyzed by Sanger sequencing consistent with homology directed repair. These findings will inform rational design of strategies for precision genome editing in plants.
Plant Cell Tissue and Organ Culture, May 26, 2016
Sugarcane is a prime crop for commercial production of bioethanol and table sugar. Genetic engine... more Sugarcane is a prime crop for commercial production of bioethanol and table sugar. Genetic engineering is complementing traditional breeding in sugarcane improvement. Costs and delays associated with regulatory approval for a transgenic event are substantial impediments to the commercialization of transgenic crops. Generation of intragenic sugarcane events carrying additional sequences from sexually compatible sorghum resembles traditional plant breeding and may facilitate regulatory approval. Acetolactate synthase (ALS), is a key enzyme in the biosynthetic pathway of branched-chain amino acids, and also a target for ALS-inhibiting herbicides including sulfonylureas and imidazolinones. In this study, we developed an intragenic minimal expression cassette encoding a mutant sorghum acetolactate synthase gene (mALS) under transcriptional control of the sorghum ubiquitin promoter and sorghum HSP 3 0 UTR. mALS was delivered into tissue cultures of sugarcane cultivar CP 88-1762 by biolistic gene transfer. A suitable selection protocol for recovery of intragenic events was developed for the herbicide bispyribac sodium. Intragenic sugarcane events were identified by PCR, and qRT-PCR analysis revealed events with different levels of mALS expression. Expression of the mALS gene in sugarcane resulted in chlorsulfuron resistance, as demonstrated by lower injury level and increased plant height following application of chlorsulfuron compared to the control cultivar. The results demonstrated that the mALS gene from sorghum supports the production of intragenic, herbicide resistant sugarcane. This is the first report of an intragenic selectable marker for sugarcane.
Plant Molecular Biology, Mar 27, 2019
Background/objective Transgenic sugarcane technology will complement breeding in the development ... more Background/objective Transgenic sugarcane technology will complement breeding in the development of advanced cultivars and will benefit the global sugar and biofuel industries. In this study, we generated a highly expressed single copy locus with sequences for site specific recombination as a landing pad for transgene stacking.
Achieving sustainable cultivation of sugarcane Volume 2, 2018
Plant Cell, Tissue and Organ Culture (PCTOC), 2016
Sugarcane is a prime crop for commercial production of bioethanol and table sugar. Genetic engine... more Sugarcane is a prime crop for commercial production of bioethanol and table sugar. Genetic engineering is complementing traditional breeding in sugarcane improvement. Costs and delays associated with regulatory approval for a transgenic event are substantial impediments to the commercialization of transgenic crops. Generation of intragenic sugarcane events carrying additional sequences from sexually compatible sorghum resembles traditional plant breeding and may facilitate regulatory approval. Acetolactate synthase (ALS), is a key enzyme in the biosynthetic pathway of branched-chain amino acids, and also a target for ALS-inhibiting herbicides including sulfonylureas and imidazolinones. In this study, we developed an intragenic minimal expression cassette encoding a mutant sorghum acetolactate synthase gene (mALS) under transcriptional control of the sorghum ubiquitin promoter and sorghum HSP 3 0 UTR. mALS was delivered into tissue cultures of sugarcane cultivar CP 88-1762 by biolistic gene transfer. A suitable selection protocol for recovery of intragenic events was developed for the herbicide bispyribac sodium. Intragenic sugarcane events were identified by PCR, and qRT-PCR analysis revealed events with different levels of mALS expression. Expression of the mALS gene in sugarcane resulted in chlorsulfuron resistance, as demonstrated by lower injury level and increased plant height following application of chlorsulfuron compared to the control cultivar. The results demonstrated that the mALS gene from sorghum supports the production of intragenic, herbicide resistant sugarcane. This is the first report of an intragenic selectable marker for sugarcane.
Crop improvement is essentially required to provide food security and better nutrition globally f... more Crop improvement is essentially required to provide food security and better nutrition globally for human beings. Plant breeders are utilizing both conventional and modern breeding techniques to increase crop production and quality. Though in the genome-sequencing era, marker-assisted selection-based breeding reduced timespan required for developing new plant variety from ~ 25 to ~ 7 years. But plant breeders are still looking for appropriate molecular method to quickly and precisely improve specific traits in plants. Recently discovered genome editing methods have revolutionized available technology for crop improvements. Genome editing is a tool of genetic engineering, which using engineered nucleases edits DNA of an organism. In editing, DNA is inserted, deleted, or replaced in the genome using engineered or designer nucleases. These nucleases create site-specific double-strand breaks (DSBs) at precise locations in the genome. The induced DSBs are repaired through more frequent n...
Biotechnology Journal, 2021
Precision genome editing by homology directed repair has tremendous potential for crop improvemen... more Precision genome editing by homology directed repair has tremendous potential for crop improvement. This study describes in planta homologous recombination mediated by CRISPR/Cas9 induced DNA double strand break in proximity to a single short (∼30 nt) homology arm. The efficiency of CRISPR/Cas9‐mediated recombination between two loxP sites was compared with Cre (Cyclization recombination enzyme) and codon‐optimized Cre‐mediated site‐specific recombination in sugarcane. A transgenic locus was generated with a selectable nptII coding sequence with terminator between two loxP sites located downstream of a constitutive promoter and acting as transcription block for the downstream promoter‐less gusA coding sequence with terminator. Recombination between the two loxP sites resulted in deletion of the transcription block and restored gus activity. This transgenic locus provided an efficient screen for identification of recombination events in sugarcane callus following biolistic delivery of Cre, codon‐optimized Cre, or the combination of sgRNA and Cas9 targeting the 5′ loxP site. The Cre codon optimized for sugarcane displayed the highest efficiency in mediating the recombination that restored gus activity followed by cre and CRISPR/Cas9. Remarkably the short region of homology of the loxP site cleaved by Cas9 (30 nt)‐mediated error‐free recombination in all 21 events from three different experiments that were analyzed by Sanger sequencing consistent with homology directed repair. These findings will inform rational design of strategies for precision genome editing in plants.
Frontiers in Genome Editing, 2021
Sugarcane is the source of 80% of the sugar and 26% of the bioethanol produced globally. However,... more Sugarcane is the source of 80% of the sugar and 26% of the bioethanol produced globally. However, its complex, highly polyploid genome (2n = 100 – 120) impedes crop improvement. Here, we report efficient and reproducible gene targeting (GT) in sugarcane, enabling precise co-editing of multiple alleles via template-mediated and homology-directed repair (HDR) of DNA double strand breaks induced by the programmable nuclease CRISPR/Cas9. The evaluation of 146 independently transformed plants from five independent experiments revealed a targeted nucleotide replacement that resulted in both targeted amino acid substitutions W574L and S653I in the acetolactate synthase (ALS) in 11 lines in addition to single, targeted amino acid substitutions W574L or S653I in 25 or 18 lines, respectively. Co-editing of up to three ALS copies/alleles that confer herbicide tolerance was confirmed by Sanger sequencing of cloned long polymerase chain reaction (PCR) amplicons. This work will enable crop improv...
PLoS ONE, 2012
Background: Salinity is a major environmental factor limiting productivity of crop plants includi... more Background: Salinity is a major environmental factor limiting productivity of crop plants including rice in which wide range of natural variability exists. Although recent evidences implicate epigenetic mechanisms for modulating the gene expression in plants under environmental stresses, epigenetic changes and their functional consequences under salinity stress in rice are underexplored. DNA methylation is one of the epigenetic mechanisms regulating gene expression in plant's responses to environmental stresses. Better understanding of epigenetic regulation of plant growth and response to environmental stresses may create novel heritable variation for crop improvement. Methodology/Principal Findings: Methylation sensitive amplification polymorphism (MSAP) technique was used to assess the effect of salt stress on extent and patterns of DNA methylation in four genotypes of rice differing in the degree of salinity tolerance. Overall, the amount of DNA methylation was more in shoot compared to root and the contribution of fully methylated loci was always more than hemi-methylated loci. Sequencing of ten randomly selected MSAP fragments indicated gene-body specific DNA methylation of retrotransposons, stress responsive genes, and chromatin modification genes, distributed on different rice chromosomes. Bisulphite sequencing and quantitative RT-PCR analysis of selected MSAP loci showed that cytosine methylation changes under salinity as well as gene expression varied with genotypes and tissue types irrespective of the level of salinity tolerance of rice genotypes. Conclusions/Significance: The gene body methylation may have an important role in regulating gene expression in organ and genotype specific manner under salinity stress. Association between salt tolerance and methylation changes observed in some cases suggested that many methylation changes are not ''directed''. The natural genetic variation for salt tolerance observed in rice germplasm may be independent of the extent and pattern of DNA methylation which may have been induced by abiotic stress followed by accumulation through the natural selection process.
Journal of Heredity, 2013
Crop Science, 2012
Seed dormancy (SD) is a key domestication trait closely related to preharvest sprouting tolerance... more Seed dormancy (SD) is a key domestication trait closely related to preharvest sprouting tolerance. Wild and weedy rices (Oryza spp.) exhibit higher degrees of seed dormancy compared to the cultivated rice. Red rice (Oryza sativa L.), a major weed in the rice growing areas of the southern United States, was used to elucidate the genetic architecture of SD. Quantitative trait loci (QTL) analysis conducted in two recombinant inbred line (RIL) populations developed from the crosses involving two rice cultivars (Bengal and Cypress) and a red rice accession (PSRR-1) revealed six to seven QTL for seed dormancy, which accounted for 49 to 52% of the total phenotypic variance. The magnitude of the QTL contribution to phenotypic variance was infl uenced by genetic backgrounds. The majority of QTL had minor effects, except the QTL linked to Rc and Sdr4. The genetic architecture for seed dormancy in U.S. red rice was distinct compared with the earlier reported weedy accessions. Four QTL were mapped onto similar positions in both populations. Both cultivars and red rice contributed alleles for increased SD. Most of the digenic epistatic interactions involved loci other than the QTL with main effects. The nucleotide polymorphisms at the Sdr4 locus could not explain the phenotypic variation for seed dormancy in our materials. The variation in SD among the rice cultivars could be attributed to segregation of minor QTL, which may be exploited to improve preharvest sprouting tolerance.
BMC Plant Biology, 2012
Background Metallothioneins (MT) are low molecular weight, cysteine rich metal binding proteins, ... more Background Metallothioneins (MT) are low molecular weight, cysteine rich metal binding proteins, found across genera and species, but their function(s) in abiotic stress tolerance are not well documented. Results We have characterized a rice MT gene, OsMT1e-P, isolated from a subtractive library generated from a stressed salinity tolerant rice genotype, Pokkali. Bioinformatics analysis of the rice genome sequence revealed that this gene belongs to a multigenic family, which consists of 13 genes with 15 protein products. OsMT1e-P is located on chromosome XI, away from the majority of other type I genes that are clustered on chromosome XII. Various members of this MT gene cluster showed a tight co-regulation pattern under several abiotic stresses. Sequence analysis revealed the presence of conserved cysteine residues in OsMT1e-P protein. Salinity stress was found to regulate the transcript abundance of OsMT1e-P in a developmental and organ specific manner. Using transgenic approach, w...
BMC Plant Biology, 2012
Background: SUMO (Small Ubiquitin related Modifier) conjugation is a post translational regulator... more Background: SUMO (Small Ubiquitin related Modifier) conjugation is a post translational regulatory process found in all eukaryotes, mediated by SUMO activating enzyme, SUMO conjugating enzyme, and SUMO ligase for the attachment of SUMO to its target protein. Although the mechanism for regulation of SUMO conjugation pathway genes under abiotic stress has been studied to certain extent, the role of SUMO conjugating enzyme in improving abiotic stress tolerance to plant is largely unexplored. Here, we have characterized a SUMO conjugating enzyme gene 'SaSce9' from a halophytic grass Spartina alterniflora and investigated its role in imparting abiotic stress tolerance. Results: SaSce9 gene encodes for a polypeptide of 162 amino acids with a molecular weight of~18 kD and isoelectric point 8.43. Amino acid sequence comparisons of SaSce9 with its orthologs from other plant species showed high degree (~85-93%) of structural conservation among each other. Complementation analysis using yeast SCE mutant, Ubc9, revealed functional conservation of SaSce9 between yeast and S. alterniflora. SaSce9 transcript was inducible by salinity, drought, cold, and exogenously supplied ABA both in leaves and roots of S. alterniflora. Constitutive overexpression of SaSce9 in Arabidopsis through Agrobacterium mediated transformation improved salinity and drought tolerance of Arabidopsis. SaSce9 overexpressing Arabidopsis plants retained more chlorophyll and proline both under salinity and drought stress. SaSce9 transgenic plants accumulated lower levels of reactive oxygen under salinity stress. Expression analysis of stress responsive genes in SaSce9 Arabidopsis plants revealed the increased expression of antioxidant genes, AtSOD and AtCAT, ion antiporter genes, AtNHX1 and AtSOS1, a gene involved in proline biosynthesis, AtP5CS, and a gene involved in ABA dependent signaling pathway, AtRD22. Conclusions: These results highlight the prospect of improving abiotic stress tolerance in plants through genetic engineering of the sumoylation pathway. The study provides evidence that the overexpression of SaSce9 in plant can improve salinity and drought stress tolerance by protecting the plant through scavenging of ROS, accumulation of an osmolyte, proline, and expression of stress responsive genes. In addition, this study demonstrates the potential of the halophyte grass S. alterniflora as a reservoir of abiotic stress related genes for crop improvement.
GCB Bioenergy
This is an open access article under the terms of the Creative Commons Attribution License, which... more This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.
Frontiers in plant science, 2018
The transgenic technology using a single gene has been widely used for crop improvement. But the ... more The transgenic technology using a single gene has been widely used for crop improvement. But the transgenic pyramiding of multiple genes, a promising alternative especially for enhancing complexly inherited abiotic stress tolerance, has received little attention. Here, we developed and evaluated transgenic rice lines with a single Salt Responsive Protein 3-1 () gene as well as pyramids with two-genes and Vacuolar H-ATPase subunit c1 () derived from a halophyte grass L. for salt tolerance at seedling, vegetative, and reproductive stages. The overexpression of this novel gene resulted in significantly better growth of with the recombinant plasmid under 600 mM NaCl stress condition compared with the control. During early seedling and vegetative stages, the single gene and pyramided transgenic rice plants showed enhanced tolerance to salt stress with minimal wilting and drying symptoms, improved shoot and root growth, and significantly higher chlorophyll content, relative water content,...
Scientific reports, Feb 1, 2018
Although flowering in rice has been extensively investigated, few studies focused on genetic inte... more Although flowering in rice has been extensively investigated, few studies focused on genetic interactions. Flowering evaluation of two recombinant inbred line (RIL) populations involving photo-insensitive rice cultivars, Bengal and Cypress, and a weedy rice accession, PSRR-1, under natural long-day (LD) conditions, revealed six to ten quantitative trait loci (QTLs) and a major QTL interaction. In addition to the validation of several previously cloned genes using an introgression lines (IL) population of PSRR-1, a few novel QTLs were also discovered. Analysis of the marker profiles of the advanced backcross lines revealed that Hd1 allele of PSRR-1 was responsible for the photoperiodic response in the near-isogenic lines (NILs) developed in both cultivar backgrounds. Based on the phenotypic and genotypic data of the NILs, and NIL mapping population and the transcript abundance of key flowering pathway genes, we conclude that Hd1 and its interaction with a novel gene other than Ghd7 p...