Matthew D. Halfhill

Transgene introgression from genetically modified crops to their wild relatives

Transgenes engineered into annual crops could be unintentionally introduced into the genomes of their free-living wild relatives. The fear is that these transgenes might persist in the environment and have negative ecological consequences. Are some crops or transgenic traits of more concern than others? Are there natural genetic barriers to minimize gene escape? Can the genetic transformation process be exploited to produce new barriers to gene flow? Questions abound, but luckily so do answers.

Transcriptional responses of Arabidopsis thaliana plants to As (V) stress

BackgroundArsenic is toxic to plants and a common environmental pollutant. There is a strong chemical similarity between arsenate [As (V)] and phosphate (Pi). Whole genome oligonucleotide microarrays were employed to investigate the transcriptional responses of Arabidopsis thaliana plants to As (V) stress.ResultsAntioxidant-related genes (i.e. coding for superoxide dismutases and peroxidases) play prominent roles in response to arsenate. The microarray experiment revealed induction of chloroplast Cu/Zn superoxide dismutase (SOD) (at2g28190), Cu/Zn SOD (at1g08830), as well as an SOD copper chaperone (at1g12520). On the other hand, Fe SODs were strongly repressed in response to As (V) stress. Non-parametric rank product statistics were used to detect differentially expressed genes. Arsenate stress resulted in the repression of numerous genes known to be induced by phosphate starvation. These observations were confirmed with qRT-PCR and SOD activity assays.ConclusionMicroarray data suggest that As (V) induces genes involved in response to oxidative stress and represses transcription of genes induced by phosphate starvation. This study implicates As (V) as a phosphate mimic in the cell by repressing genes normally induced when available phosphate is scarce. Most importantly, these data reveal that arsenate stress affects the expression of several genes with little or unknown biological functions, thereby providing new putative gene targets for future research.

Genetic load and transgenic mitigating genes in transgenic Brassica rapa (field mustard) × Brassica napus (oilseed rape) hybrid populations.

BackgroundOne theoretical explanation for the relatively poor performance of Brassica rapa (weed) × Brassica napus (crop) transgenic hybrids suggests that hybridization imparts a negative genetic load. Consequently, in hybrids genetic load could overshadow any benefits of fitness enhancing transgenes and become the limiting factor in transgenic hybrid persistence. Two types of genetic load were analyzed in this study: random/linkage-derived genetic load, and directly incorporated genetic load using a transgenic mitigation (TM) strategy. In order to measure the effects of random genetic load, hybrid productivity (seed yield and biomass) was correlated with crop- and weed-specific AFLP genomic markers. This portion of the study was designed to answer whether or not weed × transgenic crop hybrids possessing more crop genes were less competitive than hybrids containing fewer crop genes. The effects of directly incorporated genetic load (TM) were analyzed through transgene persistence data. TM strategies are proposed to decrease transgene persistence if gene flow and subsequent transgene introgression to a wild host were to occur.ResultsIn the absence of interspecific competition, transgenic weed × crop hybrids benefited from having more crop-specific alleles. There was a positive correlation between performance and number of B. napus crop-specific AFLP markers [seed yield vs. marker number (r = 0.54, P = 0.0003) and vegetative dry biomass vs. marker number (r = 0.44, P = 0.005)]. However under interspecific competition with wheat or more weed-like conditions (i.e. representing a situation where hybrid plants emerge as volunteer weeds in subsequent cropping systems), there was a positive correlation between the number of B. rapa weed-specific AFLP markers and seed yield (r = 0.70, P = 0.0001), although no such correlation was detected for vegetative biomass. When genetic load was directly incorporated into the hybrid genome, by inserting a fitness-mitigating dwarfing gene that that is beneficial for crops but deleterious for weeds (a transgene mitigation measure), there was a dramatic decrease in the number of transgenic hybrid progeny persisting in the population.ConclusionThe effects of genetic load of crop and in some situations, weed alleles might be beneficial under certain environmental conditions. However, when genetic load was directly incorporated into transgenic events, e.g., using a TM construct, the number of transgenic hybrids and persistence in weedy genomic backgrounds was significantly decreased.

Functional Genomics Analysis of Horseweed (Conyza canadensis) with Special Reference to the Evolution of Non-Target-Site Glyphosate Resistance

Abstract The evolution of glyphosate resistance in weedy species places an environmentally benign herbicide in peril. The first report of a dicot plant with evolved glyphosate resistance was horseweed, which occurred in 2001. Since then, several species have evolved glyphosate resistance and genomic information about nontarget resistance mechanisms in any of them ranges from none to little. Here, we report a study combining iGentifier transcriptome analysis, cDNA sequencing, and a heterologous microarray analysis to explore potential molecular and transcriptomic mechanisms of nontarget glyphosate resistance of horseweed. The results indicate that similar molecular mechanisms might exist for nontarget herbicide resistance across multiple resistant plants from different locations, even though resistance among these resistant plants likely evolved independently and available evidence suggests resistance has evolved at least four separate times. In addition, both the microarray and sequence analyses identified non–target-site resistance candidate genes for follow-on functional genomics analysis. Nomenclature: Glyphosate, horseweed, Conyza canadensis (L.) Cronq. ERICA

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 µg/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 µg/g leaf tissue. This study indicates that the Bt gene can persist and be stably expressed in wild B. rapa.

Laser-Induced Fluorescence Imaging and Spectroscopy of GFP Transgenic Plants

Green fluorescent protein (GFP) and other fluorescent protein bioreporters can be used to monitor transgenes in plants. GFP is a valuable marker for transgene presence and expression, but remote sensing instrumentation for stand-off detection has lagged behind fluorescent protein marker biotechnology. However, both biology and photonics are needed for the monitoring technology to be fully realized. In this paper, we describe laser-induced fluorescence imaging and laser-induced fluorescence spectroscopy of GFP-transgenic plants in ambient light towards the application of remote sensing of transgenic plants producing GFP.

Characterization of directly transformed weedy Brassica rapa and introgressed B. rapa with Bt cry1Ac and gfp genes

Crop to weed transgene flow, which could result in more competitive weed populations, is an agricultural biosafety concern. Crop Brassica napus to weedy Brassica rapa hybridization has been extensively characterized to better understand the transgene flow and its consequences. In this study, weedy accessions of B. rapa were transformed with Bacillus thuringiensis (Bt) cry1Ac- and green fluorescence protein (gfp)-coding transgenes using Agrobacterium to assess ecological performance of the wild biotype relative to introgressed hybrids in which the transgenic parent was the crop. Regenerated transgenic B. rapa events were characterized by progeny analysis, Bt protein enzyme-linked immunosorbent assay (ELISA), Southern blot analysis, and GFP expression assay. GFP expression level and Bt protein concentration were significantly different between independent transgenic B. rapa events. Similar reproductive productivity was observed in comparison between transgenic B. rapa events and B. rapa × B. napus introgressed hybrids in greenhouse and field experiments. In the greenhouse, Bt transgenic plants experienced significantly less herbivory damage from the diamondback moth (Plutella xylostella). No differences were found in the field experiment under ambient, low, herbivore pressure. Directly transformed transgenic B. rapa plants should be a helpful experimental control to better understand crop genetic load in introgressed transgenic weeds.

Green fluorescent protein quantification in whole plants.

As future biotechnology applications utilize recombinant proteins as commercial products, nondestructive assays will be necessary to determine protein concentrations accurately within plant tissues. Green fluorescent protein (GFP) has been proposed as a potential marker for the monitoring of transgenic plants and quantifying recombinant protein levels under field conditions. This chapter discusses the utility of using GFP fluorescence as an indicator of protein concentrations and the methods used to quantify GFP fluorescence in whole plant tissues. Furthermore, we discuss the accuracy and effectiveness of the portable General Fluorescence Plant Meter (GFP Meter, Opti-Sciences, Inc.) compared to a laboratory-based spectrofluorometer (Fluoro-Max2, Jobin Yvon & Glen Spectra). In whole plants, GFP fluorescence 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 over time. Younger leaves were significantly more fluorescent than older leaves on the same plant. GFP fluorescence intensity was directly correlated with the concentration of soluble protein per unit wet mass and with another genetically linked recombinant protein (Bacillus thuringiensis [Bt] cry1Ac endotoxin protein).

Transgene dispersal through pollen.

Techniques used for the transfer of novel genes into host plant genomes have created new possibilities for crop improvement. The implementation of transgenic crop species into agriculture has introduced the possibility of transgene escape into the environment via pollen dispersal. Although the movement of pollen is a critical step in transgene escape, there is currently no system to monitor transgenic pollen movement under field conditions. The development of an effective in vivo monitoring system suitable for use under field conditions is needed for research and commercial purposes so potential risks can be quantified and evaluated. This chapter describes the development of a model system using green fluorescent protein (GFP) expression in pollen as a marker to monitor pollen distribution patterns. A pollen specific promoter was used to express the GFP gene in tobacco (Nicotiana tabacum L.). GFP was visualized in pollen and growing pollen tubes using fluorescent microscopy. Furthermore, the goal of this research was to compare the dynamics of pollen movement with that of gene flow by using another method of whole plant expression of GFP to estimate out-crossing frequencies by progeny analysis. Pollen movement and gene flow were quantified under field conditions. Pollen traps were collected and screened for presence of GFP-tagged pollen using fluorescence microscopy. Progeny from wild type plants were screened with a hand held ultraviolet light for detection of the GFP phenotype.

Introgression and Persistence of an Insecticidal Transgene Can Be Monitoredby GFP

tory of policy development, in agbiotech in Australia. This will include a summary of work being done here in the area of social, legal and ethical aspects of agbiotech. The paper, as well as proposing research questions that need to be addressed in this area, will suggest that a new language, of both words and ideas, is needed to bridge discipline gaps and bring light to the debate on biotechnology.