Sample topography and position within plant body influence the detection of the intensity of green fluorescent protein fluorescence in the leaves of transgenic tobacco plants

Abstract

The effect of the type of leaf tissue selected for the study of green fluorescent protein (GFP) fluorescence intensity was investigated here using the T(1) generation of transgenic tobacco expressing the m-gfp5-ER gene. The fluorescence of GFP was detected by fluorescence binocular microscope coupled with the CCD camera and quantified by means of image analyses using the Lucia((R)) software. Mean brightness values from various leaf tissues were compared. First, an original data revealing the significant differences in the fluorescence intensity between the abaxial and adaxial surfaces are given. Stronger signal was detected on the abaxial side. Subsequently, the effect of the tissue location within the leaf surface was investigated and higher fluorescence was detected on the samples detached from leaf tips. Finally, the effect of the physiological age of leaves was studied using the in vitro clonally propagated plants. Leaves from the analogous positions within the plant body of three clones were investigated. The decrease in the fluorescence towards the plant top (youngest leaves) was observed in all studied plants. Surprisingly, the variability of the fluorescence within the clones of studied genotype was high enough to conclude, that the fluorescence of each individual is unique and affected by particular genotype and environment. Our study showed that the origin of leaf tissue selected for the GFP quantification is crucial and that the fluctuations in the fluorescence intensity should be taken into account when comparing the GFP fluorescence patterns of different plants. Moreover, the degree of fluorescence variability seems to be individually affected.

Sonication assisted Agrobacterium-mediated transformation enhances the transformation efficiency in flax (Linum usitatissimum L.)

“…An arrow indicated position of the product of expected mass. The polymerase chain reaction (PCR) following the procedure described by Hraška et al (2008) was used to confirm the presence of the gfp transgene in regenerated shoots and to exclude the simultaneous occurrence of the vir gene as a consequence of persistance of the vector bacteria after 6-9 months to SAAT. MW = 100 bp molecular weight marker; PC = positive control (pBINmGFP5-ER plasmid); 0-150 = DNA isolated of double-selected flax plants regenerated from transformation experiments based on a simple co-cultivation (0) or sonication for 50-150 s (50-150) preceeding to the co-cultivation; NC = negative control (DNA from untransformed plants) 50 s of sonication (35 kHz, 35 W) could substantially increase the efficiency of stable flax transformation (Fig.…”

“…The expression of GFP was monitored continuously up to 30 days following the SAAT with the aim of evaluating and selecting stable transformed tissues and plants. After a six to nine months period the polymerase chain reaction (PCR) following the procedure described by Hraška et al (2008) was used to confirm the presence of the gfp transgene in regenerated shoots and to exclude the simultaneous occurrence of the vir gene as a consequence of persistance of the vector bacteria.…”

Comparison of expression of three different sub-cellular targeted GFPs in transgenic Valencia sweet orange by confocal laser scanning microscopy

“…Consequently, untransformed plant tissues were used as negative controls for adjusting the parameters to ensure the absence of GFP fluorescence in nontransformed leaves. Secondly, it has been reported that expression of the marker gene is influenced by differences in leaf tissue, leaf developmental stage, and location within the plant body (Hraska et al 2008). Different cytoplasmic density of cells in young and older leaves leads to the ''dilution'' of GFP in older tissues thereby weakening the fluorescence (Omar and Grosser 2008).…”

An epifluorescent attachment improves whole-plant digital photography of Arabidopsis thaliana expressing red-shifted green fluorescent protein

“…Thus, the green channel counts observed in negative controls should be subtracted from the counts observed in GFP-expressing plants. Developmental age (Harper and Stewart 2000; Halfhill et al 2003; Hraška et al 2006, 2008), environmental growth conditions (Halfhill et al 2004) and plant species (Zhou et al 2005) can affect the efficiency of GFP fluorescence detection. Thus, controls should be physiologically identical to GFP-expressing plants in terms of genetic background, developmental ages and growth conditions.…”

“…Studies involving direct comparison of surface fluorescence and GFP content in soluble protein extracts have shown linear relationships (Blumenthal et al 1999; Harper et al 1999; Richards et al 2003; Halfhill et al 2004). Green fluorescent protein leaf surface fluorescence can be measured with probes that clip onto leaves, laboratory-based fluorescent imaging systems (Niwa et al 1999; Millwood et al 2003; Richards et al 2003; Halfhill et al 2004; Stewart 2006) and dissecting fluorescent microscopes (Zhou et al 2005; Jach 2006; Hraška et al 2008; Yambao et al 2008). Although all of these systems have specific advantages (Halfhill et al 2004), they are all expensive.…”