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

• Published in: Plant cell reports Vol. 27; no. 1; pp. 67 - 77
• Main Authors: HRASKA, Marek, HERMANOVA, Veronika, RAKOUSKY, Slavomir, CURN, Vladislav
• Format: Journal Article
• Language: English
• Published: Berlin/Heidelberg Berlin/Heidelberg : Springer-Verlag 01-01-2008; Springer-Verlag; Springer; Springer Nature B.V
• Subjects: Biological and medical sciences; Biomedical and Life Sciences; Biotechnology; Cell Biology; Cloning; Electrophoresis, Polyacrylamide Gel; Fluorescence; Fluorescence pattern; Fundamental and applied biological sciences. Psychology; gene expression; Genetic engineering; Genetic technics; Genetic Transformation and Hybridization; gfp; Green Fluorescent Proteins - genetics; Green Fluorescent Proteins - metabolism; image analysis; Leaf position; Leaves; Life Sciences; Methods. Procedures. Technologies; Microscopy, Fluorescence; Nicotiana - genetics; Nicotiana - metabolism; Plant Biochemistry; Plant Leaves - genetics; Plant Leaves - metabolism; Plant Sciences; Plant tissues; Plants; Plants, Genetically Modified - genetics; Plants, Genetically Modified - metabolism; Polymerase Chain Reaction; Proteins; Reverse Transcriptase Polymerase Chain Reaction; Transgenic animals and transgenic plants; Transgenic plants; Image analysis; Gene expression; Leaf position; Fluorescence pattern; Fluorescence; Plant leaf; Nicotiana tabacum; gfp; Image analysis, Leaf position; Dicotyledones; Topography; Angiospermae; Green fluorescent protein; Spermatophyta; Transgenic plant; Solanaceae; Detection
• ISSN: 0721-7714; 1432-203X
• DOI: 10.1007/s00299-007-0431-7

The effect of the type of leaf tissue selected for the study of green fluorescent protein (GFP) fluorescence intensity was investigated here using the T₁ 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® 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.