Reginald J. Millwood

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.

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

The level of transgene expression in crop × 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 T1 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 (BC1F1, BC2F1) 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 (BC2F2 Bulk) were generated by crossing BC2F1 individuals in the presence of a pollinating insect (Musca domestica L.). The ploidy of plants in the BC2F2 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 F1 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 BC2F2 Bulk population. Average fluorescence of each successive hybrid generation was analyzed, and homozygous canola lines and hybrid populations that contained individuals homozygous for GFP (BC2F2 Bulk) demonstrated significantly higher fluorescence than hemizygous hybrid generations (F1, BC1F1 and BC2F1). 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.

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.

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.

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.

Bacterial pathogen phytosensing in transgenic tobacco and Arabidopsis plants

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.

Computational discovery of soybean promoter cis‐regulatory elements for the construction of soybean cyst nematode‐inducible synthetic promoters

Computational methods offer great hope but limited accuracy in the prediction of functional cis-regulatory elements; improvements are needed to enable synthetic promoter design. We applied an ensemble strategy for de novo soybean cyst nematode (SCN)-inducible motif discovery among promoters of 18 co-expressed soybean genes that were selected from six reported microarray studies involving a compatible soybean-SCN interaction. A total of 116 overlapping motif regions (OMRs) were discovered bioinformatically that were identified by at least four out of seven bioinformatic tools. Using synthetic promoters, the inducibility of each OMR or motif itself was evaluated by co-localization of gain of function of an orange fluorescent protein reporter and the presence of SCN in transgenic soybean hairy roots. Among 16 OMRs detected from two experimentally confirmed SCN-inducible promoters, 11 OMRs (i.e. 68.75%) were experimentally confirmed to be SCN-inducible, leading to the discovery of 23 core motifs of 5- to 7-bp length, of which 14 are novel in plants. We found that a combination of the three best tools (i.e. SCOPE, W-AlignACE and Weeder) could detect all 23 core motifs. Thus, this strategy is a high-throughput approach for de novo motif discovery in soybean and offers great potential for novel motif discovery and synthetic promoter engineering for any plant and trait in crop biotechnology.

Introgression of bacterial leaf blight resistance and aroma genes using functional marker-assisted selection in rice (Oryza sativa L.)

Functional markers reduce the risk of false selection in marker-assisted selection (MAS), and recombination between the molecular marker and the gene of interest. The present study was conducted with the objective of combining the important basmati quality traits with resistance to bacterial leaf blight (BLB) by a combination of phenotypic selection and MAS. Screening of 29 recombinants for BLB resistant genes along with aroma (fgr) gene was performed using a suite of different markers. Twenty genotypes were found in homozygous condition for Xa21 and xa13 genes. Restriction digestion analysis with BsrI, gave nine resistant recombinants. Out of nine resistant recombinants, seven were in homozygous and two were in heterozygous conditions. Recombinants viz., IRS 5441-2-21, IRS 5441-2-79, IRS 5441-2-81, IRS 5441-2-85 and IRS 5441-2-91 possessed all the three BLB resistance genes and fgr in the homozygous condition. Recombinants with enhanced resistance to BLB, basmati quality and desirable agronomic traits were identified. This germplasm can be directly developed into commercial varieties or used as immediate potential donors of BLB resistance in basmati breeding programs.

High‐throughput functional marker assay for detection of Xa/xa and fgr genes in rice (Oryza sativa L.)

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 BC1F3 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.

Fluorescent Proteins in Transgenic Plants

Fluorescent proteins (FPs) have revolutionized many areas of biological research. In particular, plant biotechnology has been significantly advanced by harnessing the power of FPs. Aequorea victoria, green fluorescent protein (GFP), has been the most studied of the proteins, but many new FPs are discovered each year. We provide here a timely review of the current uses of FPs in whole plants and we look at the color palette of candidate proteins. Lastly, we discuss current instrumentation and methods for detection and quantification of FPs in plants.