Use of a simple semiquantitative method for appraisal of green fluorescent protein gene expression in transgenic tobacco plants

Hraška, Marek; Rakouský, S.; Kocábek, Tomáš

doi:10.1007/s10535-005-3316-z

Cited by 5 publications

(3 citation statements)

References 13 publications

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“…Very intense GFP fluorescence visible in the petioles and midribs was detected also in our previous investigations (Hraška et al 2005). In addition, also easily visible were stomatal guard cells and trichomes, which however exhibited weaker fluorescence.…”

Section: Discussionsupporting

confidence: 85%

“…Molecular characterization of transgenic plants DNA extraction and PCR detection of the presence of the mgfp5-ER gene in T 0 , T 1 and T 2 plants were performed as previously described (Hraška et al 2005), but with the different primers described below. A detailed study of transgene expression in various organs in different developmental stages, detached from T 1 plants, was performed on a mRNA level by means of reverse transcriptase PCR (RT-PCR).…”

Section: Transformation and Regeneration Of Transgenic Plantsmentioning

confidence: 99%

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Tracking of the CaMV-35S promoter performance in GFP transgenic tobacco, with a special emphasis on flowers and reproductive organs, confirmed its predominant activity in vascular tissues

Hraška

Rakouský

Čurn

2007

Plant Cell Tiss Organ Cult

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Constitutive promoters are the most common promoters used to drive the expression of various genes in monocots and dicots. Therefore, it is of intense interest to ascertain their expression patterns in various plant species, organs and during their ontogenic development. In this study, the activity of the CaMV 35S promoter in transgenic tobacco plants was assessed. In contrast to other studies, performed rather on the primary transformants (T 0 generation), here, individuals of T 1 and T 2 generations were used. The expression profiles of the CaMV 35S promoter were tracked within various plant organs and tissues using the GFP marker. Special attention was given to floral tissues for which the original data regarding the CaMV 35S expression were obtained. As expected, distinct developmental and organ/tissue specific expression patterns in a plant body were observed.

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Section: Discussionsupporting

confidence: 85%

Section: Transformation and Regeneration Of Transgenic Plantsmentioning

confidence: 99%

Tracking of the CaMV-35S promoter performance in GFP transgenic tobacco, with a special emphasis on flowers and reproductive organs, confirmed its predominant activity in vascular tissues

Hraška

Rakouský

Čurn

2007

Plant Cell Tiss Organ Cult

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“…However, very poor data are available in literature about localisation in transgenic Nicotiana spp. of stably expressed ER-retained (Hraška et al 2005) or vacuolar GFP (Di Sansebastiano et al 2005) fusion proteins, whereas several reports describe chimerical GFPs expression driven by strong tissue specific promoters (Ottenschläger et al 1999, Wright et al 2003 or in transient expression assays (Geelen et al 2002, Brandizzi et al 2003.…”

Section: Effect Of Continuous Darkness On Vacuolar Gfps Accumulationmentioning

confidence: 99%

Green fluorescent protein reveals variability in vacuoles of three plant species

et al. 2007

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Two vacuolar green fluorescent proteins (GFP) were stably inserted in Nicotiana tabacum and Nicotiana benthamiana genome, with unexpected difficulties, and compared with A. thaliana cv. Wassilewskaja transgenic plants expressing the same constructs. GFP fluorescence was strong in all tissues of A. thaliana but it was barely visible in Nicotiana. Confocal microscopy analysis revealed a variable distribution of the marker in those cells where GFP fluorescence was visible. The role of light dependent proteases was the variable pointing out more inter-species diversity. GFPs degradation was much higher in Nicotiana spp. than in A. thaliana. The version of GFP used appeared not to be a good vacuolar marker for Nicotiana differentiated tissues, although it can efficiently label vacuoles in protoplasts or calli. Nevertheless the sensitivity of the reporter protein can be used as an indicator of hidden characteristics of the plant vacuoles, revealing differences otherwise invisible. One of the markers in our system, GFP-Chi, evidenced a clear morphological difference in the vacuolar system of guard cells of the three species.

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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

et al. 2007

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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.

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Use of a simple semiquantitative method for appraisal of green fluorescent protein gene expression in transgenic tobacco plants

Cited by 5 publications

References 13 publications

Tracking of the CaMV-35S promoter performance in GFP transgenic tobacco, with a special emphasis on flowers and reproductive organs, confirmed its predominant activity in vascular tissues

Tracking of the CaMV-35S promoter performance in GFP transgenic tobacco, with a special emphasis on flowers and reproductive organs, confirmed its predominant activity in vascular tissues

Green fluorescent protein reveals variability in vacuoles of three plant species

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

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