Laser-Induced Fluorescence Imaging and Spectroscopy of GFP Transgenic Plants

Stewart, C. Neal; Millwood, Reginald J.; Halfhill, Matthew D.; Mentewab, Ayalew; Cardoza, Vinitha; Kooshki, Mitra; Capelle, Gene A.; Kyle, Kevin R.; Piaseki, David; McCrum, Gregory; Benedetto, J. Di

doi:10.1007/s10895-005-2977-5

Cited by 18 publications

(11 citation statements)

References 31 publications

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“…Nevertheless, these assays tend to be more qualitative rather than quantitative [3]. Depending on the experimental layout, different GFP detection methods are currently used, including a conventional hand-held UV lamp, fluorometers, fluorescent immunoassays, laser-induced fluorescent spectroscopy, fluorescence microscopy or confocal laser-scanning microscopy, some of which allow for quantitative GFP expression analysis [10-13]. Our results show that whole lamina imaging in an optical imaging system such as the IVIS ® Lumina II allows an accurate assessment of GFP-expression to be determined in plants, and that this methodology is highly applicable to the widely used agroinfiltration assay.…”

Section: Discussionmentioning

confidence: 99%

“…Quantification of GFP expression in plants requires either anti-GFP antibody for immunological assays or a device able to detect and measure GFP fluorescence [10]. Depending on the experimental layout, a number of different GFP imaging devices and methodologies are currently used, from conventional hand-held UV lamps, through confocal laser-scanning microscopes, some of which also allow for quantitative GFP expression analysis [11-13]. Detection and quantification of GFP is often hampered by auto-fluorescence of plant tissues, which is mainly due to chlorophyll [14].…”

Section: Introductionmentioning

confidence: 99%

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Visualization of plant viral suppressor silencing activity in intact leaf lamina by quantitative fluorescent imaging

et al. 2011

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BackgroundTransient expression of proteins in plants has become a favoured method over the production of stably transformed plants because, in addition to enabling high protein yields, it is both fast and easy to apply. An enhancement of transient protein expression can be achieved by plant virus-encoded RNA silencing suppressor proteins. Since viral suppressor proteins differ in their efficiency to enhance transient protein expression in plants, we developed a whole-leaf green fluorescent protein (GFP)-based imaging assay to quantitatively assess suppressor protein activity.ResultsIn a transient GFP-expression assay using wild-type and GFP-transgenic N. benthamiana, addition of the plant viral suppressors Beet mild yellowing virus (BMYV-IPP) P0 or Plum pox virus (PPV) HC-Pro was shown to increase fluorescent protein expression 3-4-fold, 7 days post inoculation (dpi) when compared to control plants. In contrast, in agroinfiltrated patches without suppressor activity, near complete silencing of the GFP transgene was observed in the transgenic N. benthamiana at 21 dpi. Both co-infiltrated suppressors significantly enhanced GFP expression over time, with HC-Pro co-infiltrations leading to higher short term GFP fluorescence (at 7 dpi) and P0 giving higher long term GFP fluorescence (at 21 dpi). Additionally, in contrast to HC-Pro co-infiltrations, an area of complete GFP silencing was observed at the edge of P0 co-infiltrated areas.ConclusionsFluorescence imaging of whole intact leaves proved to be an easy and effective method for spatially and quantitatively observing viral suppressor efficiency in plants. This suppressor assay demonstrates that plant viral suppressors greatly enhanced transient GFP expression, with P0 showing a more prolonged suppressor activity over time than HC-Pro. Both suppressors could prove to be ideal candidates for enhancing target protein expression in plants.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Visualization of plant viral suppressor silencing activity in intact leaf lamina by quantitative fluorescent imaging

et al. 2011

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“…The chief problem with “small scale” laboratory studies is that they are confined to tightly-controlled artificial conditions. Field experiments and radiometric models of vegetable remote sensing have the problem with long-range remote sensing is manifold: 1) ‘real-life’ systems generate data that is extremely noisy due to optical artifacts, 2) in complex environments 9 connecting robustly-measured phenomes to genes and genomes is tenuous 1,6 , 3) incomplete illumination causes partial percent-coverage and lower leaf-area-index 4) bidirectional effects, 5) sub-pixel mixing, and 6) spatial variability on the order of a square meter in the scene landscape 10 .…”

Section: Main Papermentioning

confidence: 99%

Fluorescence-based whole plant imaging and phenomics

Rigoulot

Schimel

Lee

et al. 2019

Preprint

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Summary Reverse genetics approaches have revolutionized plant biology and agriculture. Phenomics has the prospect of bridging plant phenotypes with genes, including transgenes, to transform agricultural fields1. Genetically-encoded fluorescent proteins (FPs) have transformed studies in gene expression, protein trafficking, and plant physiology. While the first instance of plant canopy imaging of green fluorescent protein (GFP) was performed over 20 years ago2, modern phenomics has largely ignored fluorescence as a transgene indicator despite the burgeoning FP color palette currently available to biologists3–5. Here we show a new platform for standoff imaging of plant canopies expressing a wide variety of FP genes in leaves. The platform, the fluorescence-inducing laser projector (FILP), uses a low-noise camera to image a scene illuminated by compact diode lasers of various colors and emission filters to phenotype transgenic plants expressing multiple constitutive or inducible FPs. Of the 20 FPs screened, we selected the top performing candidates for standoff phenomics at ≥ 3 m using FILP in a laboratory-based laser range. Included in demonstrated applications is the performance of an osmotic stress-inducible synthetic promoter selected from a high throughput library screen. While FILP has unprecedented versatility as a laboratory platform, we envisage future iterations of the system for use in automated greenhouse or even drone-fielded versions of the platform for crop screening.

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“…Along with GFP and other FP bioreporters, LIF can be used to monitor transgenes in plants [45] and to detect plant diseases and plant stress [46]. Some 3-D imaging systems use confocal microscopy to obtain fluorescence signals within a small biological sample volume [47].…”

Section: Laser-induced Atomic and Molecular Fluorescencementioning

confidence: 99%