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C N Stewart

Publications and source records attributed to C N Stewart.

At least 19 recordsLinked to original sources
PubMed2006

Correlated expression of gfp and Bt cry1Ac gene facilitates quantification of transgenic hybridization between Brassicas.

Gene flow from transgenic oilseed rape (BRASSICA NAPUS) might not be avoidable, thus, it is important to detect and quantify hybridization events with its relatives in real time. Data are presented showing the correlation between genetically linked green fluorescent protein (GFP) with BACILLUS THURINGIENSIS (Bt) CRY1AC gene expression in hybrids formed between transgenic B. NAPUS "Westar" and a wild Chinese accession of wild mustard (B. JUNCEA) and hybridization between transgenic B. NAPUS and a conspecific Chinese landrace oilseed rape. Hybrids were obtained either by spontaneous hybridization in the field or by hand-crossing in a greenhouse. In all cases, transgenic hybrids were selected by GFP fluorescence among seedlings originating from seeds harvested from B. JUNCEA and the Chinese oilseed rape plants. Transgenicity was confirmed by PCR detection of transgenes. GFP fluorescence was easily and rapidly detected in the hybrids under greenhouse and field conditions. Results showed that both GFP fluorescence and Bt protein synthesis decreased as either plant or leaf aged, and GFP fluorescence intensity was closely correlated with Bt protein concentration during the entire vegetative lifetime in hybrids. These findings allow the use of GFP fluorescence as an accurate tool to detect gene-flow in time in the field and to conveniently estimate BT CRY1AC expression in hybrids on-the-plant.

Bacillus thuringiensis Toxins↗
PubMed2005

Laboratory studies of the effects of reduced prey choice caused by Bt plants on a predatory insect.

Crops transformed to express Bacillus thuringiensis (Bt) toxins can cause close to 100% mortality of certain target pest species. This study assessed the effect of target pest reduction on the predatory insect Chrysoperla carnea (Stephens) in the presence of alternative prey. Numbers of lacewings recovered from Bt oilseed rape (cultivar Oscar, event O52) did not differ significantly from numbers of lacewings recovered from conventional oilseed rape in cage experiments with the target pest Plutella xylostella (Linnaeus) and the non-target pest Myzus persicae (Sulzer) when aphid densities were high. However, significantly fewer lacewings were recovered from Bt plants as aphid densities were lowered. Lacewing weights were not affected by plant type.

Animals↗
PubMed2004

Stable Bacillus thuringiensis (Bt) toxin content in interspecific F1 and backcross populations of wild Brassica rapa after Bt gene transfer.

Stable expression of a transgene may lead to increased fitness for wild plants after acquiring the transgene via crop-weed hybridization. Here, we investigate the stability of Bt toxin content in wild Brassica rapa acquiring the Bt gene from Bt Brassica napus. The Bt toxin content in nine Bt-expressing B. napus lines was 0.80-1.70 micro g/g leaf tissue throughout the growing season. These nine lines were crossed with three accessions of wild B. rapa and the Bt gene was successfully transferred to interspecific hybrids (F1) and successive backcross generations (BC1 to BC4). The Bt toxin level in F1 and BC progenies containing the Bt gene remained at 0.90-3.10 micro g/g leaf tissue. This study indicates that the Bt gene can persist and be stably expressed in wild B. rapa.

Bacillus thuringiensis↗
PubMed2003

Additive transgene expression and genetic introgression in multiple green-fluorescent protein transgenic crop x weed hybrid generations.

The level of transgene expression in crop x weed hybrids and the degree to which crop-specific genes are integrated into hybrid populations are important factors in assessing the potential ecological and agricultural risks of gene flow associated with genetic engineering. The average transgene zygosity and genetic structure of transgenic hybrid populations change with the progression of generations, and the green fluorescent protein (GFP) transgene is an ideal marker to quantify transgene expression in advancing populations. The homozygous T(1) single-locus insert GFP/ Bacillus thuringiensis (Bt) transgenic canola ( Brassica napus, cv Westar) with two copies of the transgene fluoresced twice as much as hemizygous individuals with only one copy of the transgene. These data indicate that the expression of the GFP gene was additive, and fluorescence could be used to determine zygosity status. Several hybrid generations (BC(1)F(1), BC(2)F(1)) were produced by backcrossing various GFP/Bt transgenic canola ( B. napus, cv Westar) and birdseed rape ( Brassica rapa) hybrid generations onto B. rapa. Intercrossed generations (BC(2)F(2) Bulk) were generated by crossing BC(2)F(1) individuals in the presence of a pollinating insect ( Musca domestica L.). The ploidy of plants in the BC(2)F(2) Bulk hybrid generation was identical to the weedy parental species, B. rapa. AFLP analysis was used to quantify the degree of B. napus introgression into multiple backcross hybrid generations with B. rapa. The F(1) hybrid generations contained 95-97% of the B. napus-specific AFLP markers, and each successive backcross generation demonstrated a reduction of markers resulting in the 15-29% presence in the BC(2)F(2) Bulk population. Average fluorescence of each successive hybrid generation was analyzed, and homozygous canola lines and hybrid populations that contained individuals homozygous for GFP (BC(2)F(2) Bulk) demonstrated significantly higher fluorescence than hemizygous hybrid generations (F(1), BC(1)F(1) and BC(2)F(1)). These data demonstrate that the formation of homozygous individuals within hybrid populations increases the average level of transgene expression as generations progress. This phenomenon must be considered in the development of risk-management strategies.

Crops, Agricultural↗
PubMed2003

Spatial and temporal patterns of green fluorescent protein (GFP) fluorescence during leaf canopy development in transgenic oilseed rape, Brassica napus L.

The green fluorescent protein (GFP) holds promise as a field-level transgene marker. One obstacle to the use of GFP is fluorescence variability observed within leaf canopies. In growth chamber and field experiments, GFP fluorescence in transgenic oilseed rape ( Brassica napus) was shown to be variable at each leaf position over time and among different leaves on the same plant. A leaf had its highest GFP fluorescence after emergence and, subsequently, its fluorescence intensity decreased. GFP fluorescence intensity was directly correlated with the concentration of soluble protein. The concentration of the genetically linked recombinant Bacillus thuringiensis (Bt) cry1Ac endotoxin protein also was examined, and GFP fluorescence was positively correlated with Bt throughout development. The results show that GFP can be used as an accurate transgene marker but that aspects of plant developmental should be taken into account when interpreting fluorescence measurements.

Bacillus thuringiensis Toxins↗
PubMed2003

Quantitative GFP fluorescence as an indicator of recombinant protein synthesis in transgenic plants.

The utility of green fluorescent protein (GFP) for biological research is evident. A fluorescence-based method was developed to quantify GFP levels in transgenic plants and protein extracts. Fluorescence intensity was linear with increasing levels of GFP over a range that encompasses transgene expression in plants by the cauliflower mosaic virus 35S promoter. Standard curves were used to estimate GFP concentration in planta and in protein extracts. These values were consistent with ELISA measurements of GFP in protein extracts from transgenic plants, indicating that the technique is a reliable measure of recombinant GFP expression. The levels of in planta GFP expression in both homozygous and hemizygous plants was then estimated. Homozygous transgenic plants expressed twice the amount of GFP than hemizygous plants, suggesting additive transgene expression. This methodology may be useful to simplify the characterization of transgene expression in plants.

Enzyme-Linked Immunosorbent Assay↗
PubMed2003

Hybridization between transgenic Brassica napus L. and its wild relatives: Brassica rapa L., Raphanus raphanistrum L., Sinapis arvensis L., and Erucastrum gallicum (Willd.) O.E. Schulz.

The frequency of gene flow from Brassica napus L. (canola) to four wild relatives, Brassica rapa L., Raphanus raphanistrum L., Sinapis arvensis L. and Erucastrum gallicum (Willd.) O.E. Schulz, was assessed in greenhouse and/or field experiments, and actual rates measured in commercial fields in Canada. Various marker systems were used to detect hybrid individuals: herbicide resistance traits (HR), green fluorescent protein marker (GFP), species-specific amplified fragment length polymorphisms (AFLPs) and ploidy level. Hybridization between B. rapa and B. napus occurred in two field experiments (frequency approximately 7%) and in wild populations in commercial fields (approximately 13.6%). The higher frequency in commercial fields was most likely due to greater distance between B. rapa plants. All F(1) hybrids were morphologically similar to B. rapa, had B. napus- and B. rapa-specific AFLP markers and were triploid (AAC, 2n=29 chromosomes). They had reduced pollen viability (about 55%) and segregated for both self-incompatible and self-compatible individuals (the latter being a B. napus trait). In contrast, gene flow between R. raphanistrum and B. napus was very rare. A single R. raphanistrum x B. napus F1 hybrid was detected in 32,821 seedlings from the HR B. napus field experiment. The hybrid was morphologically similar to R. raphanistrum except for the presence of valves, a B. napus trait, in the distorted seed pods. It had a genomic structure consistent with the fusion of an unreduced gamete of R. raphanistrum and a reduced gamete of B. napus (RrRrAC, 2n=37), both B. napus- and R. raphanistrum-specific AFLP markers, and had <1% pollen viability. No hybrids were detected in the greenhouse experiments (1,534 seedlings), the GFP field experiment (4,059 seedlings) or in commercial fields in Québec and Alberta (22,114 seedlings). No S. arvensis or E. gallicum x B. napus hybrids were detected (42,828 and 21,841 seedlings, respectively) from commercial fields in Saskatchewan. These findings suggest that the probability of gene flow from transgenic B. napus to R. raphanistrum, S. arvensis or E. gallicum is very low (<2-5 x 10(-5)). However, transgenes can disperse in the environment via wild B. rapa in eastern Canada and possibly via commercial B. rapa volunteers in western Canada.

Brassicaceae↗
PubMed2003

Instrumentation and methodology for quantifying GFP fluorescence in intact plant organs.

The General Fluorescence Plant Meter (GFP-Meter) is a portable spectrofluorometer that utilizes a fiber-optic cable and a leaf clip to gather spectrofluorescence data. In contrast to traditional analytical systems, this instrument allows for the rapid detection and fluorescence measurement of proteins under field conditions with no damage to plant tissue. Here we discuss the methodology of gathering and standardizing spectrofluorescence data from tobacco and canola plants expressing GFP. Furthermore, we demonstrate the accuracy and effectiveness of the GFP-Meter. We first compared GFP fluorescence measurements taken by the GFP-Meter to those taken by a standard laboratory-based spectrofluorometer, the FluoroMax-2. Spectrofluorescence measurements were taken from the same location on intact leaves. When these measurements were tested by simple linear regression analysis, we found that there was a positive functional relationship between instruments. Finally, to exhibit that the GFP-Meter recorded accurate measurements over a span of time, we completed a time-course analysis of GFP fluorescence measurements. We found that only initial measurements were accurate; however, subsequent measurements could be used for qualitative purposes.

Brassica↗
PubMed2002

Increased Agrobacterium-mediated transformation and rooting efficiencies in canola (Brassica napus L.) from hypocotyl segment explants.

An efficient protocol for the production of transgenic Brassica napus cv. Westar plants was developed by optimizing two important parameters: preconditioning time and co-cultivation time. Agrobacterium tumefaciens-mediated transformation was performed using hypocotyls as explant tissue. Two variants of a green fluorescent protein (GFP)-encoding gene--mGFP5-ER and eGFP--both under the constitutive expression of the cauliflower mosaic virus 35S promoter, were used for the experiments. Optimizing the preconditioning time to 72 h and co-cultivation time with Agrobacterium to 48 h provided the increase in the transformation efficiency from a baseline of 4% to 25%. With mGFP5-ER, the transformation rate was 17% and with eGFP it was 25%. Transgenic shoots were selected on 200 mg/l kanamycin. Rooting efficiency was 100% on half-strength Murashige and Skoog medium with 10 g/l sucrose and 0.5 mg/l indole butyric acid in the presence of kanamycin.

Agrobacterium tumefaciens↗
PubMed2001

The utility of green fluorescent protein in transgenic plants.

The green fluorescent protein (GFP) from the jellyfish Aequorea victoria has proven to be a powerful tool in plant genetic transformation studies. This paper reviews the history and the progression of the expression of GFP variants in transgenic plants. The distinguishing features of the most useful GFPs, such as those including the S65T chromophore mutation and those with dual excitation peaks, are discussed. The review also focuses on the utility of GFP as a visual selectable marker in aiding the plant transformation process; GFP has been more important in monocot transformation compared with dicot transformation. Finally, the potential utility of new fluorescent proteins is speculated upon.

Animals↗
PubMed2000

Sodium detection during the water absorption response in Rana pipiens.

Although taste in vertebrates is typically associated with specialized receptors in the lingual epithelium, Hoff and Hillyard reported that the toad, Bufo punctatus, is able to "taste" sodium with the abdominal skin. This was reflected in a differential behavioral response to hypertonic NaCl. The present study tests for the presence of such abdominal chemoreceptors in the frog Rana pipiens. The experiment was a five-condition design in which frogs were placed on filter paper saturated with: deionized water, 250 mM NaCl, 350 mM NaCl, 12.9 microM amiloride, or 350 mM NaCl + 12.9 microM amiloride. The time that the frogs remained on the test substrate before moving to a surface of deionized water was recorded. It was necessary to dehydrate the frogs to 80% of their body weight to elicit a behavioral response to the NaCl whereas dehydration to 90% of their body weight has been reported effective in Bufo punctatus. The frogs displayed significantly shorter mean times to move on both concentrations of NaCl compared to deionized water, with the shortest times occurring when 350 mM NaCl was used. Amiloride alone did not have an effect upon times to move to deionized water, but did significantly reduce the response to 350 mM NaCl. Movement to amiloride + 350 mM NaCl did not differ significantly from that to deionized water. The results indicate that Rana pipiens, like Bufo punctatus, have epithelial chemoreceptors for the detection of NaCl on hydrated surfaces and that these receptors, like those of mammals, are amiloride sensitive.

Abdomen↗
PubMed2000

Transgenic plants and biosafety: science, misconceptions and public perceptions.

One usually thinks of plant biology as a non-controversial topic, but the concerns raised over the biosafety of genetically modified (GM) plants have reached disproportionate levels relative to the actual risks. While the technology of changing the genome of plants has been gradually refined and increasingly implemented, the commercialization of GM crops has exploded. Today's commercialized transgenic plants have been produced using Agrobacterium tumefaciens-mediated transformation or gene gun-mediated transformation. Recently, incremental improvements of biotechnologies, such as the use of green fluorescent protein (GFP) as a selectable marker, have been developed. Non-transformation genetic modification technologies such as chimeraplasty will be increasingly used to more precisely modify germplasm. In spite of the increasing knowledge about genetic modification of plants, concerns over ecological and food biosafety have escalated beyond scientific rationality. While several risks associated with GM crops and foods have been identified, the popular press, spurred by colorful protest groups, has left the general public with a sense of imminent danger. Reviewed here are the risks that are currently under research. Ecological biosafety research has identified potential risks associated with certain crop/transgene combinations, such as intra- and interspecific transgene flow, persistence and the consequences of transgenes in unintended hosts. Resistance management strategies for insect resistance transgenes and non-target effects of these genes have also been studied. Food biosafety research has focused on transgenic product toxicity and allergenicity. However, an estimated 3.5 x 10(12) transgenic plants have been grown in the U.S. in the past 12 years, with over two trillion being grown in 1999 and 2000 alone. These large numbers and the absence of any negative reports of compromised biosafety indicate that genetic modification by biotechnology poses no immediate or significant risks and that resulting food products from GM crops are as safe as foods from conventional varieties. We are increasingly convinced that scientists have a duty to conduct objective research and to effectively communicate the results--especially those pertaining to the relative risks and potential benefits--to scientists first and then to the public. All stakeholders in the technology need more effective dialogues to better understand risks and benefits of adopting or not adopting agricultural biotechnologies.

Culture Techniques↗
PubMed1999

Green fluorescent protein as a marker for expression of a second gene in transgenic plants.

The use of transgenic crops has generated concerns about transgene movement to unintended hosts and the associated ecological consequences. Moreover, the in-field monitoring of transgene expression is of practical concern (e.g., the underexpression of an herbicide tolerance gene in crop plants that are due to be sprayed with herbicide). A solution to these potential problems is to monitor the presence and expression of an agronomically important gene by linking it to a marker gene, such as GFP. Here we show that GFP fluorescence can indicate expression of the Bacillus thuringiensus cry1Ac gene when co-introduced into tobacco and oilseed rape, as demonstrated by insect bioassays and western blot analysis. Furthermore we conducted two seasons of field experiments to characterize the performance of three different GFP genes in transgenic tobacco. The best gene tested was mGFP5er, a mutagenized GFP gene that is targeted to the endoplasmic reticulum. We also demonstrated that host plants synthesizing GFP in the field suffered no fitness costs.

Bacillus thuringiensis↗
PubMed1997

Applications of green fluorescent protein in plants.

Green fluorescent protein (GFP) is increasingly being used in plant biology from the cellular level to whole plant level. At the cellular level, GFP is being used as an in vivo reporter to assess frequency of transient and stable transformation. GFP has also proven to be an invaluable tool in monitoring trafficking and subcellular localization of protein. At the organ level and up, many exciting applications are rapidly emerging. The development of brighter GFP mutants with more robust folding properties has enabled better macroscopic visualization of GFP in whole leaves and plants. One interesting example has been the use of GFP to monitor virus movement in and among whole plants. GFP is also emerging as a powerful tool to monitor transgene movement and transgenic plants in the field. In a proof-of-concept study, tobacco was transformed with a modified version of the GFP gene controlled by a constitutive (35S) promoter. GFP expression in progeny plants ranged from 0% to 0.5%, and approximately 0.1% GFP was the minimal amount needed for unambiguous macroscopic detection. GFP is the first truly in vivo reporter system useful in whole plants, and we project its usefulness will increase even further as better forms of GFP genes become available.

Biotechnology↗