41 results on '"C. Neal Stewart"'
Search Results
2. Engineered gamma radiation phytosensors for environmental monitoring
- Author
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Robert G. Sears, Stephen B. Rigoulot, Alessandro Occhialini, Britany Morgan, Tayebeh Kakeshpour, Holly Brabazon, Caitlin N. Barnes, Erin M. Seaberry, Brianna Jacobs, Chandler Brown, Yongil Yang, Tayler M. Schimel, Scott C. Lenaghan, and C. Neal Stewart
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Plant Science ,Agronomy and Crop Science ,Biotechnology - Published
- 2023
3. Plant Biotechnology and Genetics: Principles, Techniques, and Applications
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C. Neal Stewart, C. Neal Stewart and C. Neal Stewart, C. Neal Stewart
- Published
- 2016
4. Kinase‐dead mutation: A novel strategy for improving soybean resistance to soybean cyst nematode Heterodera glycines
- Author
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Logan Baldwin, Tarek Hewezi, Tracy Hawk, Sarbottam Piya, C. Neal Stewart, Bhoomi Patel, and John Hollis Rice
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Cysts ,Abiotic stress ,Kinase ,Soybean cyst nematode ,food and beverages ,Soil Science ,Mercury ,Plant Science ,Biology ,biology.organism_classification ,Phenotype ,Cell biology ,Mutation ,Gene expression ,Animals ,Gene co-expression network ,Soybeans ,Tylenchoidea ,Protein Kinases ,Agronomy and Crop Science ,Molecular Biology ,Gene ,Cellular localization ,Plant Diseases - Abstract
Protein kinases phosphorylate proteins for functional changes and are involved in nearly all cellular processes, thereby regulating almost all aspects of plant growth and development, and responses to biotic and abiotic stresses. We generated two independent co-expression networks of soybean genes using control and stress response gene expression data and identified 392 differentially highly interconnected kinase hub genes among the two networks. Of these 392 kinases, 90 genes were identified as "syncytium highly connected hubs", potentially essential for activating kinase signalling pathways in the nematode feeding site. Overexpression of wild-type coding sequences of five syncytium highly connected kinase hub genes using transgenic soybean hairy roots enhanced plant susceptibility to soybean cyst nematode (SCN; Heterodera glycines) Hg Type 0 (race 3). In contrast, overexpression of kinase-dead variants of these five syncytium kinase hub genes significantly enhanced soybean resistance to SCN. Additionally, three of the five tested kinase hub genes enhanced soybean resistance to SCN Hg Type 1.2.5.7 (race 2), highlighting the potential of the kinase-dead approach to generate effective and durable resistance against a wide range of SCN Hg types. Subcellular localization analysis revealed that kinase-dead mutations do not alter protein cellular localization, confirming the structure-function of the kinase-inactive variants in producing loss-of-function phenotypes causing significant decrease in nematode susceptibility. Because many protein kinases are highly conserved and are involved in plant responses to various biotic and abiotic stresses, our approach of identifying kinase hub genes and their inactivation using kinase-dead mutation could be translated for biotic and abiotic stress tolerance.
- Published
- 2021
5. Mini‐synplastomes for plastid genetic engineering
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Scott C. Lenaghan, Agnieszka Anna Piatek, Henry Daniell, Alexander C. Pfotenhauer, Alessandro Occhialini, Li Li, Andrew J. Lail, C. Neal Stewart, Cristiano Piasecki, Stacee A. Harbison, and Jason N. Burris
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homologous recombination ,Plant Science ,Computational biology ,Biology ,Minicircle ,Genome ,Metabolic engineering ,Synthetic biology ,episomal replication ,Plastids ,Transgenes ,Plastid ,Research Articles ,Solanum tuberosum ,fungi ,food and beverages ,Plants ,plastome ,Chloroplast ,plastid engineering ,Metabolic Engineering ,Chloroplast DNA ,Synthetic Biology ,Genetic Engineering ,Homologous recombination ,small synthetic plastome ‘mini‐synplastome’ ,Agronomy and Crop Science ,Research Article ,Biotechnology - Abstract
Summary In the age of synthetic biology, plastid engineering requires a nimble platform to introduce novel synthetic circuits in plants. While effective for integrating relatively small constructs into the plastome, plastid engineering via homologous recombination of transgenes is over 30 years old. Here we show the design–build–test of a novel synthetic genome structure that does not disturb the native plastome: the ‘mini‐synplastome’. The mini‐synplastome was inspired by dinoflagellate plastome organization, which is comprised of numerous minicircles residing in the plastid instead of a single organellar genome molecule. The first mini‐synplastome in plants was developed in vitro to meet the following criteria: (i) episomal replication in plastids; (ii) facile cloning; (iii) predictable transgene expression in plastids; (iv) non‐integration of vector sequences into the endogenous plastome; and (v) autonomous persistence in the plant over generations in the absence of exogenous selection pressure. Mini‐synplastomes are anticipated to revolutionize chloroplast biotechnology, enable facile marker‐free plastid engineering, and provide an unparalleled platform for one‐step metabolic engineering in plants.
- Published
- 2021
6. Research Ethics for Scientists: A Companion for Students
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C. Neal Stewart and C. Neal Stewart
- Published
- 2011
7. Cell‐Type‐Specific Proteomics Analysis of a Small Number of Plant Cells by Integrating Laser Capture Microdissection with a Nanodroplet Sample Processing Platform
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Yiran Liang, Ying Zhu, C. Neal Stewart, Eduardo Blumwald, Vimal Kumar Balasubramanian, Amir H. Ahkami, Ryan T. Kelly, Ljiljana Paša-Tolić, Samuel O. Purvine, and William B. Chrisler
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Proteomics ,Tissue Fixation ,General Immunology and Microbiology ,Protein digestion ,Computer science ,General Neuroscience ,Sample processing ,Microfluidics ,Health Informatics ,Laser Capture Microdissection ,Plant cell ,Mass spectrometry ,General Biochemistry, Genetics and Molecular Biology ,Medical Laboratory Technology ,Plant Cells ,Sample preparation ,General Pharmacology, Toxicology and Pharmaceutics ,Biological system ,Chromatography, Liquid ,Laser capture microdissection - Abstract
Plant organs and tissues contain multiple cell types, which are well organized in 3-dimensional structure to efficiently perform physiological functions such as homeostasis and response to environmental perturbation and pathogen infection. It is critically important to perform molecular measurements at the cell-type-specific level to discover mechanisms and unique features of cell populations that govern differentiation and respond to external perturbations. Although mass spectrometry-based proteomics has been demonstrated as an enabling discovery tool for studying plant physiology, conventional approaches require millions of cells to generate robust biological conclusions. Such requirements mask the cell-to-cell heterogeneities and limit the comprehensive profiling of plant proteins at spatially resolved and cell-type-specific resolutions. This article describes a recently developed proteomics workflow for studying a small number of plant cells by integrating laser capture microdissection, microfluidic nanodroplet-based sample preparation, and ultrasensitive liquid chromatography-mass spectrometry. Using poplar as a model tree species, we provide detailed protocols, including plant leaf and root tissue harvest, sample preparation, cryosectioning, laser microdissection, protein digestion, mass spectrometry measurement, and data analysis. We show that the workflow enables the precise identification and quantification of thousands of proteins from hundreds of isolated plant root and leaf cells. © 2021 Wiley Periodicals LLC. Basic Protocol 1: Plant tissue fixation and embedding Support Protocol 1: Preparation of 2.5% CMC solution Support Protocol 2: Slow freezing of CMC blocks to avoid crack development in the block Basic Protocol 2: Preparation of cryosections Alternate Protocol: Using a vacuum manifold to dehydrate the cryosection slides (primarily for root tissues) Basic Protocol 3: Laser capture microdissection of specific types of plant cells Basic Protocol 4: Nanodroplet-based sample preparation for ultrasensitive proteomic analysis Support Protocol 3: Fabrication of nanowell chips Basic Protocol 5: Liquid chromatography and mass spectrometry.
- Published
- 2021
8. Loss of C‐genome‐specific markers during transgene introgression from Brassica napus to wild Brassica juncea
- Author
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Wei Wei, Zheng-jun Guan, Bao-cheng Shen, C. Neal Stewart, and Zhi-Xi Tang
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Biosafety ,Transgene ,Botany ,Brassica ,Introgression ,Plant Science ,Biology ,biology.organism_classification ,Genome ,Ecology, Evolution, Behavior and Systematics - Published
- 2020
9. Transgenic miR156 switchgrass in the field: growth, recalcitrance and rust susceptibility
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Holly L. Baxter, C. Neal Stewart, Jace Natzke, Chunxiang Fu, Brian H. Davison, Zeng-Yu Wang, Geoffrey B. Turner, Steven D. Brown, Mitra Mazarei, Mark F. Davis, Jiqing Gou, Alexandru Dumitrache, Robert W. Sykes, and Miguel Rodriguez
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0301 basic medicine ,Perennial plant ,Field experiment ,Transgene ,switchgrass ,Biomass ,Growing season ,transgene regulation ,Plant Science ,Plant disease resistance ,Biology ,Panicum ,complex mixtures ,03 medical and health sciences ,Gene Expression Regulation, Plant ,Bioenergy ,microRNA156 ,bioconfinement ,Research Articles ,Plant Proteins ,flowering ,biomass ,food and beverages ,Plants, Genetically Modified ,MicroRNAs ,030104 developmental biology ,Agronomy ,Biofuel ,Agronomy and Crop Science ,Research Article ,Biotechnology - Abstract
Summary Sustainable utilization of lignocellulosic perennial grass feedstocks will be enabled by high biomass production and optimized cell wall chemistry for efficient conversion into biofuels. MicroRNAs are regulatory elements that modulate the expression of genes involved in various biological functions in plants, including growth and development. In greenhouse studies, overexpressing a microRNA (miR156) gene in switchgrass had dramatic effects on plant architecture and flowering, which appeared to be driven by transgene expression levels. High expressing lines were extremely dwarfed, whereas low and moderate expressing lines had higher biomass yields, improved sugar release, and delayed flowering. Four lines with moderate or low miR156 overexpression from the prior greenhouse study were selected for a field experiment to assess the relationship between miR156 expression and biomass production over three years. We also analyzed important bioenergy feedstock traits such as flowering, disease resistance, cell wall chemistry, and biofuel production. Phenotypes of the transgenic lines were inconsistent between the greenhouse and the field as well as among different field growing seasons. One low expressing transgenic line consistently produced more biomass (25-56%) than the control across all three seasons, which translated to the production of 30% more biofuel per plant during the final season. The other three transgenic lines produced less biomass than the control by the final season, and the two lines with moderate expression levels also exhibited altered disease susceptibilities. Results of this study emphasize the importance of performing multi-year field studies for plants with altered regulatory transgenes that target plant growth and development. This article is protected by copyright. All rights reserved.
- Published
- 2017
10. The evolutionary history of ferns inferred from 25 low‐copy nuclear genes
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Xiao Sun, Li Chen, Sean W. Graham, Douglas E. Soltis, Michael K. Deyholos, Dennis W. Stevenson, Erin M. Sigel, Lisa Pokorny, Kathleen M. Pryer, Dylan O. Burge, Fay-Wei Li, Lisa DeGironimo, Shane W. Shaw, Xiaofeng Wei, C. Neal Stewart, Claude W. dePamphilis, Petra Korall, Anders Larsson, Markus Ruhsam, Layne Huiet, Gane Ka-Shu Wong, Tao Chen, and Carl J. Rothfels
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Nuclear gene ,DNA, Plant ,Molecular Sequence Data ,Gene Dosage ,Plant Science ,Coalescent theory ,Phylogenetics ,Phylogenomics ,Genetics ,Phylogeny ,Ecology, Evolution, Behavior and Systematics ,Base Sequence ,Models, Genetic ,biology ,Phylogenetic tree ,Sequence Analysis, DNA ,biology.organism_classification ,Biological Evolution ,Sister group ,Genetic Loci ,Evolutionary biology ,Ferns ,Outgroup ,Fern ,Transcriptome ,Sequence Alignment - Abstract
3 PREMISE OF THE STUDY: Understanding fern (monilophyte) phylogeny and its evolutionary timescale is critical for broad investigations of the evolution of land plants, and for providing the point of comparison necessary for studying the evolution of the fern sister group, seed plants. Molecular phylogenetic investigations have revolutionized our understanding of fern phylogeny, however, to date, these studies have relied almost exclusively on plastid data. METHODS: Here we take a curated phylogenomics approach to infer the fibroad fern phylogeny from multiple nuclear loci, by combining broad taxon sampling (73 ferns and 12 outgroup species) with focused character sampling (25 loci comprising 35 877 bp), along with rigorous alignment, orthology inference and model selection. KEY RESULTS: Our phylogeny corroborates some earlier inferences and provides novel insights; in particular, we fi nd strong support for Equisetales as sister to the rest of ferns, Marattiales as sister to leptosporangiate ferns, and Dennstaedtiaceae as sister to the eupolypods. Our divergence-time analyses reveal that divergences among the extant fern orders all occurred prior to ~200 MYA. Finally, our species-tree inferences are congruent with analyses of concat- enated data, but generally with lower support. Those cases where species-tree support values are higher than expected involve relationships that have been supported by smaller plastid datasets, suggesting that deep coalescence may be reducing support from the concatenated nuclear data. CONCLUSIONS: Our study demonstrates the utility of a curated phylogenomics approach to inferring fern phylogeny, and highlights the need to consider underlying data characteristics, along with data quantity, in phylogenetic studies.
- Published
- 2015
11. Transgenic switchgrass (Panicum virgatumL.) biomass is increased by overexpression of switchgrass sucrose synthase (PvSUS1)
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Mitra Mazarei, Charleson R. Poovaiah, Mark F. Davis, Robert W. Sykes, Geoffrey B. Turner, Stephen R. Decker, and C. Neal Stewart
- Subjects
Sucrose ,Biomass ,Panicum ,complex mixtures ,Applied Microbiology and Biotechnology ,chemistry.chemical_compound ,Bioenergy ,Botany ,Lignin ,Cellulose ,Plant Proteins ,biology ,fungi ,food and beverages ,General Medicine ,Plants, Genetically Modified ,biology.organism_classification ,chemistry ,Glucosyltransferases ,Biofuel ,Biofuels ,biology.protein ,Molecular Medicine ,Sucrose synthase ,Panicum virgatum ,Biotechnology - Abstract
Sucrose synthase (SUS) converts sucrose and uridine di-phosphate (UDP) into UDP-glucose and fructose. UDP-glucose is used by the cellulose synthase to produce cellulose for cell wall biosynthesis. For lignocellulosic feedstocks such as switchgrass, the manipulation of cell walls to decrease lignin content is needed to reduce recalcitrance of conversion of biomass into biofuels. Of perhaps equal importance for bioenergy feedstocks is increasing biomass. Four SUS genes were identified in switchgrass. Each gene contained 14 or 15 introns. PvSUS1 was expressed ubiquitously in the tissues tested. PvSUS2 and PvSUS6 were highly expressed in internodes and roots, respectively. PvSUS4 was expressed in low levels in the tissues tested. Transgenic switchgrass plants overexpressing PvSUS1 had increases in plant height by up to 37%, biomass by up to 13.6%, and tiller number by up to 79% compared to control plants. The lignin content was increased in all lines, while the sugar release efficiency was decreased in PvSUS1-overexpressing transgenic switchgrass plants. For switchgrass and other bioenergy feedstocks, the overexpression of SUS1 genes might be a feasible strategy to increase both plant biomass and cellulose content, and to stack with other genes to increase biofuel production per land area cultivated.
- Published
- 2014
12. ‘Fukusensor:’ a genetically engineered plant for reporting DNA damage in response to gamma radiation
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Sara M. Allen, C. Neal Stewart, Yanhui Peng, and Reginald J. Millwood
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DNA damage ,Green Fluorescent Proteins ,Mutant ,Arabidopsis ,Plant Science ,Genetically modified crops ,medicine.disease_cause ,Green fluorescent protein ,Genes, Reporter ,medicine ,Arabidopsis thaliana ,Gene ,Genetics ,Mutation ,biology ,fungi ,food and beverages ,Plants, Genetically Modified ,biology.organism_classification ,Cell biology ,Gamma Rays ,Genetic Engineering ,Agronomy and Crop Science ,DNA Damage ,Biotechnology - Abstract
Transgenic plants can be designed to be 'phytosensors' for detection of environmental contaminants and pathogens. In this study, we describe the design and testing of a radiation phytosensor in the form of green fluorescence protein (GFP)-transgenic Arabidopsis plant utilizing a DNA repair deficiency mutant background as a host. Mutant lines of Arabidopsis AtATM (At3g48190), which are hypersensitive to gamma irradiation, were used to generate stable GFP transgenic plants in which a gfp gene was under the control of a strong constitutive CaMV 35S promoter. Mutant and nonmutant genetic background transgenic plants were treated with 0, 1, 5, 10 and 100 Gy radiation doses, respectively, using a Co-60 source. After 1 week, the GFP expression levels were drastically reduced in young leaves of mutant background plants (treated by 10 and 100 Gy), whereas there were scant visible differences in the fluorescence of the nonmutant background plants. These early results indicate that transgenic plants could serve in a relevant sensor system to report radiation dose and the biological effects to organisms in response to radionuclide contamination.
- Published
- 2014
13. Narrow terahertz attenuation signaturesin Bacillus thuringiensis
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Weidong Zhang, C. Neal Stewart, Elliott R. Brown, Leamon Viveros, and Kellie P. Burris
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Materials science ,biology ,Spectrometer ,business.industry ,Terahertz radiation ,Attenuation ,General Engineering ,Analytical chemistry ,General Physics and Astronomy ,General Chemistry ,biology.organism_classification ,General Biochemistry, Genetics and Molecular Biology ,Photomixing ,Bacillus thuringiensis ,Optoelectronics ,General Materials Science ,Vibrational resonance ,business ,Spectroscopy ,Absorption (electromagnetic radiation) - Abstract
Terahertz absorption signatures from culture-cultivated Bacillus thuringiensis were measured with a THz photomixing spectrometer operating from 400 to 1200 GHz. We observe two distinct signatures centered at ∼955 and 1015 GHz, and attribute them to the optically coupled particle vibrational resonance (surface phonon-polariton) of Bacillus spores. This demonstrates the potential of the THz attenuation signatures as “fingerprints” for label-free biomolecular detection. (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim)
- Published
- 2013
14. Gene use restriction technologies for transgenic plant bioconfinement
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C. Neal Stewart, Reginald J. Millwood, and Yi Sang
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business.industry ,Transgene ,Plant Science ,Plants ,Biology ,Plants, Genetically Modified ,Biotechnology ,Genetic Techniques ,Risk analysis (engineering) ,Transgenes ,State of the science ,business ,Agronomy and Crop Science ,Gene - Abstract
Summary The advances of modern plant technologies, especially genetically modified crops, are considered to be a substantial benefit to agriculture and society. However, so-called transgene escape remains and is of environmental and regulatory concern. Genetic use restriction technologies (GURTs) provide a possible solution to prevent transgene dispersal. Although GURTs were originally developed as a way for intellectual property protection (IPP), we believe their maximum benefit could be in the prevention of gene flow, that is, bioconfinement. This review describes the underlying signal transduction and components necessary to implement any GURT system. Furthermore, we review the similarities and differences between IPP- and bioconfinement-oriented GURTs, discuss the GURTs’ design for impeding transgene escape and summarize recent advances. Lastly, we go beyond the state of the science to speculate on regulatory and ecological effects of implementing GURTs for bioconfinement.
- Published
- 2013
15. Bacterial pathogen phytosensing in transgenic tobacco andArabidopsisplants
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Wusheng Liu, C. Neal Stewart, Mary R. Rudis, Michael H. Fethe, Gisele Schoene, Jason N. Burris, Yanhui Peng, Mitra Mazarei, and Reginald J. Millwood
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Crops, Agricultural ,Transgene ,Green Fluorescent Proteins ,Arabidopsis ,Cyclopentanes ,Plant Science ,Genetically modified crops ,Genes, Plant ,Microbiology ,chemistry.chemical_compound ,Plant Growth Regulators ,Genes, Reporter ,Tobacco ,Botany ,Oxylipins ,Regulatory Elements, Transcriptional ,Transgenes ,Promoter Regions, Genetic ,Pathogen ,Disease Resistance ,Plant Diseases ,Methyl jasmonate ,biology ,fungi ,food and beverages ,Gene Expression Regulation, Bacterial ,Ethylenes ,Plants, Genetically Modified ,biology.organism_classification ,Plant disease ,chemistry ,Host-Pathogen Interactions ,Salicylic Acid ,Agronomy and Crop Science ,Bacteria ,Salicylic acid ,Biotechnology - Abstract
Summary Plants are subject to attack by a wide range of phytopathogens. Current pathogen detection methods and technologies are largely constrained to those occurring post-symptomatically. Recent efforts were made to generate plant sentinels (phytosensors) that can be used for sensing and reporting pathogen contamination in crops. Engineered phytosensors indicating the presence of plant pathogens as early-warning sentinels potentially have tremendous utility as wide-area detectors. We previously showed that synthetic promoters containing pathogen and/ or defence signalling inducible cis-acting regulatory elements (RE) fused to a fluorescent protein (FP) reporter could detect phytopathogenic bacteria in a transient phytosensing system. Here, we further advanced this phytosensing system by developing stable transgenic tobacco and Arabidopsis plants containing candidate constructs. The inducibility of each synthetic promoter was examined in response to biotic (bacterial pathogens) or chemical (plant signal molecules salicylic acid, ethylene and methyl jasmonate) treatments using stably transgenic plants. The treated plants were visualized using epifluorescence microscopy and quantified using spectrofluorometry for FP synthesis upon induction. Time-course analyses of FP synthesis showed that both transgenic tobacco and Arabidopsis plants were capable to respond in predictable ways to pathogen and chemical treatments. These results provide insights into the potential applications of transgenic plants as phytosensors and the implementation of emerging technologies for monitoring plant disease outbreaks in agricultural fields.
- Published
- 2012
16. Assessment and Detection of Gene Flow
- Author
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Charles Kwit, Wei Wei, Hong S. Moon, Reginald J. Millwood, and C. Neal Stewart
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Genetics ,Computational biology ,Biology ,Gene flow - Published
- 2012
17. Overexpression of miR156 in switchgrass ( Panicum virgatum L.) results in various morphological alterations and leads to improved biomass production
- Author
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Ramanjulu Sunkar, Yuhong Tang, Ji-Yi Zhang, Hui Shen, Chunxiang Fu, Jessica Matts, David G. J. Mann, Zeng-Yu Wang, Jennifer J. Wolf, Chuanen Zhou, and C. Neal Stewart
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0106 biological sciences ,biofuel crop ,Apical dominance ,Carbohydrates ,Biomass ,Plant Science ,Genetically modified crops ,Genes, Plant ,Panicum ,01 natural sciences ,7. Clean energy ,transgenic switchgrass ,03 medical and health sciences ,Gene Expression Regulation, Plant ,Bioenergy ,RNA, Messenger ,Research Articles ,Plant Proteins ,030304 developmental biology ,miR156 ,2. Zero hunger ,0303 health sciences ,biomass ,microRNA ,biology ,Hydrolysis ,food and beverages ,Oryza ,15. Life on land ,Herbaceous plant ,Plants, Genetically Modified ,biology.organism_classification ,Energy crop ,MicroRNAs ,Panicum virgatum ,Solubility ,Agronomy ,Agronomy and Crop Science ,010606 plant biology & botany ,Biotechnology - Abstract
Summary Switchgrass (Panicum virgatum L.) has been developed into a dedicated herbaceous bioenergy crop. Biomass yield is a major target trait for genetic improvement of switchgrass. microRNAs have emerged as a prominent class of gene regulatory factors that has the potential to improve complex traits such as biomass yield. A miR156b precursor was overexpressed in switchgrass. The effects of miR156 overexpression on SQUAMOSA PROMOTER BINDING PROTEIN LIKE (SPL) genes were revealed by microarray and quantitative RT-PCR analyses. Morphological alterations, biomass yield, saccharification efficiency and forage digestibility of the transgenic plants were characterized. miR156 controls apical dominance and floral transition in switchgrass by suppressing its target SPL genes. Relatively low levels of miR156 overexpression were sufficient to increase biomass yield while producing plants with normal flowering time. Moderate levels of miR156 led to improved biomass but the plants were nonflowering. These two groups of plants produced 58%‐101% more biomass yield compared with the control. However, high miR156 levels resulted in severely stunted growth. The degree of morphological alterations of the transgenic switchgrass depends on miR156 level. Compared with floral transition, a lower miR156 level is required to disrupt apical dominance. The improvement in biomass yield was mainly because of the increase in tiller number. Targeted overexpression of miR156 also improved solubilized sugar yield and forage digestibility, and offered an effective approach for transgene containment.
- Published
- 2012
18. Gateway-compatible vectors for high-throughput gene functional analysis in switchgrass (Panicum virgatum L.) and other monocot species
- Author
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Laura L. Abercrombie, Peter R. LaFayette, Zachary R. King, Richard A. Dixon, Mathew C. Halter, Mitra Mazarei, Holly L. Baxter, Hui Shen, David G. J. Mann, Charleson R. Poovaiah, Wayne A. Parrott, and C. Neal Stewart
- Subjects
Agrobacterium ,business.industry ,fungi ,food and beverages ,Plant Science ,Genetically modified crops ,Biology ,biology.organism_classification ,Gateway cassette ,Biotechnology ,Transformation (genetics) ,Cellulosic ethanol ,Bioenergy ,Panicum virgatum ,Vector (molecular biology) ,business ,Agronomy and Crop Science - Abstract
Summary Switchgrass (Panicum virgatum L.) is a C4 perennial grass and has been identified as a potential bioenergy crop for cellulosic ethanol because of its rapid growth rate, nutrient use efficiency and widespread distribution throughout North America. The improvement of bioenergy feedstocks is needed to make cellulosic ethanol economically feasible, and genetic engineering of switchgrass is a promising approach towards this goal. A crucial component of creating transgenic switchgrass is having the capability of transforming the explants with DNA sequences of interest using vector constructs. However, there are limited options with the monocot plant vectors currently available. With this in mind, a versatile set of Gateway- compatible destination vectors (termed pANIC) was constructed to be used in monocot plants for transgenic crop improvement. The pANIC vectors can be used for transgene overexpres- sion or RNAi-mediated gene suppression. The pANIC vector set includes vectors that can be utilized for particle bombardment or Agrobacterium-mediated transformation. All the vectors contain (i) a Gateway cassette for overexpression or silencing of the target sequence, (ii) a plant selection cassette and (iii) a visual reporter cassette. The pANIC vector set was function- ally validated in switchgrass and rice and allows for high-throughput screening of sequences of interest in other monocot species as well.
- Published
- 2011
19. Finding the Perfect Mentor
- Author
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C. Neal Stewart
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business.industry ,Pedagogy ,Medicine ,business - Published
- 2011
20. Plagiarise and Perish
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C. Neal Stewart
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Library science ,Psychology - Published
- 2011
21. Research Misconduct: Falsification and Whistleblowing
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C. Neal Stewart
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Political science ,Engineering ethics ,Scientific misconduct - Published
- 2011
22. Research Ethics: The Best Ethical Practices Produce the Best Science
- Author
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C. Neal Stewart
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Research ethics ,Political science ,Meta-ethics ,Engineering ethics - Published
- 2011
23. Authorship: Who's an Author on a Scientific Paper and Why
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C. Neal Stewart
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Political science ,Social science - Published
- 2011
24. Grant Proposals: Ethics and Success Intertwined
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C. Neal Stewart
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business.industry ,Political science ,Public administration ,Public relations ,business - Published
- 2011
25. How Corrupt is Science?
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C. Neal Stewart
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business.industry ,Political science ,Public administration ,Public relations ,business - Published
- 2011
26. Peer Review and The Ethics of Privileged Information
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C. Neal Stewart
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business.industry ,Political science ,Preprint ,Public relations ,business - Published
- 2011
27. Data and Data Management: The Ethics of Data
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C. Neal Stewart
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World Wide Web ,Data curation ,business.industry ,Computer science ,Data management ,Data management plan ,business ,Enterprise data management ,Data science - Published
- 2011
28. What Kind of Research Science World Do We Want?
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C. Neal Stewart
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Political science ,Engineering ethics ,Science education - Published
- 2011
29. Becoming the Perfect Mentor
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C. Neal Stewart
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business.industry ,Medicine ,Public relations ,business - Published
- 2011
30. High-throughput functional marker assay for detection of Xa/xa and fgr genes in rice (Oryza sativa L.)
- Author
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Sujata Agarwal, Romesh Kumar Salgotra, Reginald J. Millwood, and C. Neal Stewart
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Genetic Markers ,Clinical Biochemistry ,Pcr cloning ,Biology ,Genes, Plant ,Polymerase Chain Reaction ,Biochemistry ,Analytical Chemistry ,chemistry.chemical_compound ,Genotype ,Multiplex polymerase chain reaction ,Allele ,Gene ,Plant Proteins ,Genetics ,Polymorphism, Genetic ,Oryza sativa ,Electrophoresis, Capillary ,Receptor Protein-Tyrosine Kinases ,food and beverages ,Oryza ,Sequence Analysis, DNA ,Marker-assisted selection ,High-Throughput Screening Assays ,chemistry ,Agarose ,Microsatellite Repeats - Abstract
We apply CE for high-throughput analysis of functional markers for marker-assisted selection in rice. The accuracy, throughput and reproducibility of CE analysis for sequence-tagged site (STS) and simple sequence repeat (SSR) markers for bacterial blight resistance and aroma genes are demonstrated by using a CE system. Multiplex PCR products displayed well-differentiated allelic variants using different STS and SSR markers for identification of xa13, Xa21 and fgr genes using the CE system compared to 1.2% agarose gel images. Moreover, consumption of PCR product is much less in the CE system compared to traditional agarose gel systems. Sample consumption is less than 0.1 μL per analysis, thereby conserving samples for further downstream analysis. Out of 29 genotypes in BC(1)F(3) generation, 16 plants were found homozygous for all the three genes, viz., xa13, Xa21 and fgr. These homozygous lines can be used as potential donors in rice breeding programmes.
- Published
- 2011
31. Overexpression of epsps transgene in weedy rice: insufficient evidence to support speculations about biosafety
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Christopher J. Leaver, L Val Giddings, Jens C. Streibig, Vivian Moses, Albert J. Fischer, Amy Lawton-Rauh, Nilda R. Burgos, Aldo Merotto, Klaus Ammann, C. Neal Stewart, Jonathan Gressel, and Anthony J. Trewavas
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Oryza sativa ,Herbicides ,Physiology ,business.industry ,Transgene ,Oryza ,Shikimic Acid ,Plant Science ,Biology ,Plants, Genetically Modified ,Biotechnology ,Biosafety ,Botany ,Genetic Fitness ,Transgenes ,3-Phosphoshikimate 1-Carboxyvinyltransferase ,business ,Weedy rice - Published
- 2014
32. Characterization of the horseweed (Conyza canadensis) transcriptome using GS-FLX 454 pyrosequencing and its application for expression analysis of candidate non-target herbicide resistance genes
- Author
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Joshua S. Yuan, R. Douglas Sammons, C. Neal Stewart, Yanhui Peng, Patrick J. Tranel, Laura L. G. Abercrombie, and Chance W. Riggins
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Genetics ,Contig ,biology ,Accession number (library science) ,General Medicine ,biology.organism_classification ,DNA sequencing ,Transcriptome ,Plant protein ,Insect Science ,Conyza canadensis ,Pyrosequencing ,Weed ,Agronomy and Crop Science - Abstract
BACKGROUND:Thedenovotranscriptomesequencingofaweedy plantusingGS-FLX454technologiesisreported.Horseweed (Conyza canadensis L.) was the first broadleaf weed to evolve glyphosate resistance in agriculture, and also is the most widely distributedglyphosate-resistantweedintheUnitedStatesandtheworld.However,availablesequencedataforthisspeciesare scant.Thetranscriptomicsequenceshouldbeusefulforgenediscovery,andtohelpelucidatethenon-target-basedglyphosate resistance mechanismand the genomic basis of weediness. RESULTS: Sequencing experiments yielded 411962 raw reads, an average read length of 233 bp and a total dataset of 95.8 Mb (NCBI accession number SRA010952). After trimming and quality control, 379152 high-quality sequences were retained and assembled into contigs. The assembly resulted in 31783 unique transcripts, including 16102 contigs and 15681 singletons. The average coverage depth for each contig and each nucleotide position was 22-fold and 12-fold respectively. A total of 16306 unique sequences were annotated by searching a custom plant protein database. The utility of the transcriptome data was demonstratedby further explorationof ABC transporters,which were previously hypothesized to play a role in non-target glyphosate resistance. Real-time RT-PCR primers were designed from the transcriptome data, which made it possible to assess expression patterns of 17 ABC transporters from resistant and susceptible horseweed accessions from Tennessee, with and without glyphosate treatment. CONCLUSION:Theseresultsshow thatGS-FLX454sequencingisapowerful andcost-effective platform forthedevelopmentof functional genomic tools for a weed species. c � 2010 Society of Chemical Industry Supportinginformationmaybefoundintheonlineversionofthisarticle.
- Published
- 2010
33. Selection of Bioassay Method Influences Detection of Annual Bluegrass Resistance to Mitotic-Inhibiting Herbicides
- Author
-
Matthew A. Cutulle, John C. Sorochan, J. Scott McElroy, Reginald W. Millwood, and C. Neal Stewart
- Subjects
Pesticide resistance ,biology ,Ecotype ,biology.organism_classification ,Hydroponics ,Pendimethalin ,chemistry.chemical_compound ,Agronomy ,chemistry ,Bioassay ,Poa annua ,Dithiopyr ,Weed ,Agronomy and Crop Science - Abstract
Dinitroaniline-resistant annual bluegrass (Poa annua L.) has been reported in several states; however, there are no standardized screening methods for detecting resistance. Research was conducted to evaluate screening techniques (Murashige and Skoog [MS] media, filter paper, hydroponics, and soil based) to detect herbicide resistance to dithiopyr, prodiamine, and pendimethalin in a suspected resistant ecotype of annual bluegrass from Chattanooga, TN (Chattanooga). A senstitive ecotype from Fresno, CA (Control) was also tested. All the bioassays were able to diagnose the ecotype from Chattanooga as resistant to prodiamine and pendimethalin. However, the degree of resistance was highly variable between bioassays. In hydroponics, the amount of prodiamine required to inhibit Chattanooga growth by 50% was 26 times more than Control. Comparatively, in MS media the amount of prodiamine required to inhibit Chattanooga growth by 50% was 80 times more than Control. Minor dithiopyr resistance from the Chattanooga ecotype was detected by the hydroponics, filter-paper and soil-based bioassays. Hydroponics provided the most rapid diagnosis of resistance, accessing resistance for a mature plant in 10 d. The MS-media bioassay had the least amount of confounding variables. These findings highlight the potential variation in results that can occur in mitotic-inhibiting herbicide resistance detection simply on the basis of how plant samples are assayed.
- Published
- 2009
34. Effects of elevated carbon dioxide and ozone on volatile terpenoid emissions and multitrophic communication of transgenic insecticidal oilseed rape ( Brassica napus )
- Author
-
Delia M. Pinto, Anne Nissinen, Jarmo K. Holopainen, Sari Himanen, Guy M. Poppy, C. Neal Stewart, and Anne-Marja Nerg
- Subjects
Herbivore ,Diamondback moth ,Agronomy ,biology ,Physiology ,Bacillus thuringiensis ,Brassica ,Plutella ,Plant Science ,Genetically modified crops ,Cotesia vestalis ,biology.organism_classification ,Terpenoid - Abstract
Summary • Does transgenically incorporated insect resistance affect constitutive and herbivore-inducible terpenoid emissions and multitrophic communication under elevated atmospheric CO2 or ozone (O3)? This study aimed to clarify the possible interactions between allocation to direct defences (Bacillus thuringiensis (Bt) toxin production) and that to endogenous indirect defences under future climatic conditions. • Terpenoid emissions were measured from vegetative-stage non-Bt and Bt Brassica napus grown in growth chambers under control or doubled CO2, and control (filtered air) or 100 ppb O3. The olfactometric orientation of Cotesia vestalis, an endoparasitoid of the herbivorous diamondback moth (Plutella xylostella), was assessed under the corresponding CO2 and O3 concentrations. • The response of terpenoid emission to CO2 or O3 elevations was equivalent for Bt and non-Bt plants, but lower target herbivory reduced herbivore-inducible emissions from Bt plants. Elevated CO2 increased emissions of most terpenoids, whereas O3 reduced total terpenoid emissions. Cotesia vestalis orientated to host-damaged plants independent of plant type or CO2 concentration. Under elevated O3, host-damaged non-Bt plants attracted 75% of the parasitoids, but only 36.8% of parasitoids orientated to host-damaged Bt plants. • Elevated O3 has the potential to perturb specialized food-web communication in Bt crops.
- Published
- 2008
35. Interactions of elevated carbon dioxide and temperature with aphid feeding on transgenic oilseed rape: Are Bacillus thuringiensis (Bt) plants more susceptible to nontarget herbivores in future climate?
- Author
-
Anne-Marja Nerg, Jarmo K. Holopainen, Anne Nissinen, C. Neal Stewart, Sari Himanen, Wen-Xia Dong, and Guy M. Poppy
- Subjects
Global and Planetary Change ,Aphid ,Ecology ,biology ,fungi ,Brassica ,food and beverages ,biology.organism_classification ,chemistry.chemical_compound ,Nutrient ,chemistry ,Agronomy ,Cry1Ac ,Brevicoryne brassicae ,Chlorophyll ,Bacillus thuringiensis ,Environmental Chemistry ,Myzus persicae ,General Environmental Science - Abstract
Climate change factors such as elevated carbon dioxide (CO2) and temperature typically affect carbon (C) and nitrogen (N) dynamics of crop plants and the performance of insect herbivores. Insect-resistant transgenic plants invest some nutrients to the production of specific toxic proteins [i.e. endotoxins from Bacillus thuringiensis (Bt)], which could alter the C–N balance of these plants, especially under changed abiotic conditions. Aphids are nonsusceptible to Lepidoptera-targeted Bt Cry1Ac toxin and they typically show response to abiotic conditions, and here we sought to discover whether they might perform differently on compositionally changed Bt oilseed rape. Bt oilseed rape had increased N content in the leaves coupled with reduced total C compared with its nontransgenic counterpart, but in general the C : N responses of both plant types to elevated CO2 and temperature were similar. Elevated CO2 decreased N content and increased C : N ratio of both plant types. Elevated temperature increased C and N contents, total chlorophyll and carotenoid concentrations under ambient CO2, but decreased these under elevated CO2. In addition, soluble sugars were increased and starch decreased by elevated temperature under ambient but not under elevated CO2, whereas photosynthesis was decreased in plants grown under elevated temperature in both CO2 levels. Myzus persicae, a generalist aphid species, responded directly to elevated temperature with reduced developmental time and decreased adult and progeny weights, whereas the development of the Brassica specialist Brevicoryne brassicae was less affected. Feeding by M. persicae resulted in an increase in the N content of oilseed rape leaves under ambient CO2, indicating the potential of herbivore feeding itself to cause allocation changes. The aphids performed equally well on both plant types despite the differences between C–N ratios of Bt and non-Bt oilseed rape, revealing the absence of plant composition-related effects on these pests under elevated CO2, elevated temperature or combined elevated CO2 and temperature conditions.
- Published
- 2008
36. Statistical methods for efficiency adjusted real-time PCR quantification
- Author
-
Joshua S. Yuan, Donglin Wang, and C. Neal Stewart
- Subjects
Models, Statistical ,Models, Genetic ,Reverse Transcriptase Polymerase Chain Reaction ,business.industry ,Computer science ,Design of experiments ,Linear model ,Statistical model ,DNA ,General Medicine ,computer.software_genre ,Applied Microbiology and Biotechnology ,Set (abstract data type) ,Software ,Data Interpretation, Statistical ,Molecular Medicine ,Computer Simulation ,Data mining ,business ,Linear combination ,computer ,Equivalence (measure theory) ,Algorithms ,Statistical hypothesis testing - Abstract
The statistical treatment for hypothesis testing using real-time PCR data is a challenge for quantification of gene expression. One has to consider two key factors in precise statistical analysis of real-time PCR data: a well-defined statistical model and the integration of amplification efficiency (AE) into the model. Previous publications in real-time PCR data analysis often fall short in integrating the AE into the model. Novel, user-friendly, and universal AE-integrated statistical methods were developed for real-time PCR data analysis with four goals. First, we addressed the definition of AE, introduced the concept of efficiency-adjusted Delta Delta Ct, and developed a general mathematical method for its calculation. Second, we developed several linear combination approaches for the estimation of efficiency adjusted Delta Delta Ct and statistical significance for hypothesis testing based on different mathematical formulae and experimental designs. Statistical methods were also adopted to estimate the AE and its equivalence among the samples. A weighted Delta Delta Ct method was introduced to analyze the data with multiple internal controls. Third, we implemented the linear models with SAS programs and analyzed a set of data for each model. In order to allow other researchers to use and compare different approaches, SAS programs are included in the Supporting Information. Fourth, the results from analysis of different statistical models were compared and discussed. Our results underline the differences between the efficiency adjusted Delta Delta Ct methods and previously published methods, thereby better identifying and controlling the source of errors introduced by real-time PCR data analysis.
- Published
- 2008
37. An Arabidopsis thaliana ABC transporter that confers kanamycin resistance in transgenic plants does not endow resistance to Escherichia coli
- Author
-
Ayalew Mentewab, C. Neal Stewart, Steven Ripp, and Kellie P. Burris
- Subjects
Genetics ,Kanamycin Resistance ,fungi ,food and beverages ,Bioengineering ,ATP-binding cassette transporter ,Genetically modified crops ,Biology ,medicine.disease_cause ,biology.organism_classification ,Applied Microbiology and Biotechnology ,Biochemistry ,Subcloning ,Horizontal gene transfer ,medicine ,Arabidopsis thaliana ,Escherichia coli ,Bacteria ,Biotechnology - Abstract
Concerns have been raised about potential horizontal gene transfer (HGT) of antibiotic resistance markers (ARMs) from transgenic plants to bacteria of medical and environmental importance. All ARMs used in transgenic plants have been bacterial in origin, but it has been recently shown that an Arabidopsis thaliana ABC transporter, Atwbc19, confers kanamycin resistance when overexpressed in transgenic plants. Atwbc19 was evaluated for its ability to transfer kanamycin resistance to Escherichia coli, a kanamycin-sensitive model bacterium, under simulated HGT, staged by subcloning Atwbc19 under the control of a bacterial promoter, genetically transforming to kanamycin-sensitive bacteria, and assessing if resistance was conferred as compared with bacteria harbouring nptII, the standard kanamycin resistance gene used to produce transgenic plants. NptII provided much greater resistance than Atwbc19 and was significantly different from the no-plasmid control at low concentrations. Atwbc19 was not significantly different from the no-plasmid control at higher concentrations. Even though HGT risks are considered low with nptII, Atwbc19 should have even lower risks, as its encoded protein is possibly mistargeted in bacteria.
- Published
- 2007
38. Expression of green fluorescent protein in pollen of oilseed rape (Brassica napus L.) and its utility for assessing pollen movement in the field
- Author
-
Laura C. Hudson, Matthew D. Halfhill, C. Neal Stewart, Hong S. Moon, and Reginald J. Millwood
- Subjects
Transgene ,Green Fluorescent Proteins ,Brassica ,Molecular Probe Techniques ,Genetically modified crops ,Biology ,medicine.disease_cause ,Applied Microbiology and Biotechnology ,Green fluorescent protein ,Gene flow ,Motion ,Pollen ,Botany ,otorhinolaryngologic diseases ,medicine ,Brassica napus ,fungi ,food and beverages ,General Medicine ,biology.organism_classification ,Recombinant Proteins ,Transformation (genetics) ,Spectrometry, Fluorescence ,Seeds ,Molecular Medicine ,Fluorescent tag - Abstract
Transgene movement via pollen is an important component of gene flow from transgenic plants. Here, we present proof-of-concept studies that demonstrate the monitoring of short distant movement of pollen expressing a genetically encoded fluorescent tag in oilseed rape (Brassica napus L. cv. Westar). Transgenic oilseed rape plants were produced using Agrobacterium-mediated transformation method with the pBINDC1 construct containing a green fluorescent protein (GFP) variant, mGFP5-ER, under the control of the pollen-specific LAT59 promoter from tomato. Transgenic pollen was differentiated from non-transgenic pollen in vivo by a unique spectral signature, and was shown to be an effective tool to monitor pollen movement in the greenhouse and field. GFP-tagged pollen also served as a practical marker to determine the zygosity of plants. In a greenhouse pollen flow study, more pollen was captured at closer distances from the source plant plot with consistent wind generated by a fan. Under field conditions, GFP transgenic pollen grains were detected up to a distance of 15 m, the farthest distance from source plants assayed. GFP-tagged pollen was easily distinguishable from non-transgenic pollen using an epifluorescence microscope.
- Published
- 2006
39. Intraspecific Competition of an Insect‐Resistant Transgenic Canola in Seed Mixtures
- Author
-
Paul L. Raymer, Suresh Ramachandran, John N. All, G. David Buntin, and C. Neal Stewart
- Subjects
food.ingredient ,Diamondback moth ,biology ,fungi ,Brassica ,food and beverages ,Plutella ,Genetically modified crops ,medicine.disease_cause ,biology.organism_classification ,Diamondback ,food ,Agronomy ,parasitic diseases ,Infestation ,medicine ,Cultivar ,Canola ,Agronomy and Crop Science - Abstract
Seed mixtures are recommended as a strategy to minimize or avoid having insects develop resistance to insect-resistant transgenic crops. The objective of this study was to evaluate a canola, Brassica napus L., transgenic for a Bacillus thuringiensis cry1Ac gene for its resistance against diamondback moth, Plutella xylostella L., and for its competitive ability with nontransgenic canola in seed mixtures. Transgenic and nontransgenic canola were planted either as pure stands or in mixtures of 75:25, 50:50, and 25:75 in plastic trays in greenhouse experiments or in field experiments at three locations during the 1996 through 1998 field seasons. The trays and plots were either infested with diamondback moth neonates or left without any insect infestation. In diamondback moth-infested treatments, transgenic plants had low levels of damage both as a pure stand and in mixtures. Nontransgenic plants in diamondback moth-infested trays and plots suffered high levels of defoliation and produced less biomass and seed yield compared with transgenic plants. Relative crowding coefficient (RCC), a measure of competition between the two plant types, ranged from 0.6 to 1.1 in plots where there was no diamondback moth infestation and 1.1 to 12.8 in plots where there was diamondback moth infestation. No competitive advantage was observed for either plant type in seed mixtures when there was no diamondback moth infestation. Transgenic canola because of its high level of resistance was competitively superior in seed mixtures when there was diamondback moth infestation.
- Published
- 2000
40. Greenhouse and field evaluations of transgenic canola against diamondback moth, Plutella xylostella, and corn earworm, Helicoverpa zea
- Author
-
Suresh Ramachandran, Paul L. Raymer, John N. All, C. Neal Stewart, and G. David Buntin
- Subjects
food.ingredient ,Diamondback moth ,biology ,fungi ,Brassica ,food and beverages ,Plutella ,biology.organism_classification ,medicine.disease_cause ,food ,Agronomy ,Plutellidae ,Insect Science ,Infestation ,medicine ,Helicoverpa zea ,Cultivar ,Canola ,Ecology, Evolution, Behavior and Systematics - Abstract
Canola (Brassica napusL.) cultivars Oscar and Westar, engineered with a Bacillus thuringiensis(Bt) cryIA(c) gene, were evaluated for resistance to lepidopterous pests, diamondback moth, Plutella xylostella L. (Plutellidae) and corn earworm, Helicoverpa zea(Boddie) (Noctuidae) in greenhouse and field conditions. In greenhouse preference assays conducted at vegetative and flowering plant stages, transgenic plants recorded very low levels of damage. A 100% diamondback moth mortality and90% corn earworm mortality were obtained on transgenic plants in greenhouse antibiosis assays. The surviving corn earworm larvae on transgenic plants had reduced head capsule width and body weight. Mortality of diamondback moth and corn earworm were 100% and95%, respectively, at different growth stages (seedling, vegetative, bolting, and flowering) on the transgenic plants in greenhouse tests. In field tests conducted during 1995‐1997, plots were artificially infested with neonates of diamondback moth or corn earworm or left for natural infestation. Transgenic plants in all the treatments were highly resistant to diamondback moth and corn earworm larvae and had very low levels of defoliation. Plots infested with diamondback moth larvae had greater damage in both seasons as compared with corn earworm infested plots and plots under natural infestation. After exposure to defoliators, transgenic plants usually had higher final plant stand and produced more pods and seeds than non-transgenic plants. Diamondback moth injury caused the most pronounced difference in plant stand and pod and seed number between transgenic and non-transgenic plants. Our results suggest that transgenic canola could be used for effective management of diamondback moth and corn earworm on canola.
- Published
- 1998
41. Plant Biotechnology and Genetics
- Author
-
C. Neal Stewart
- Subjects
Genetics ,business.industry ,Plant genetics ,Genetically modified crops ,Biology ,business ,Biotechnology - Abstract
Plant biotechnology and genetics : , Plant biotechnology and genetics : , مرکز فناوری اطلاعات و اطلاع رسانی کشاورزی
- Published
- 2007
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