African Journal of Biotechnology Vol. 4 (12), pp. 1372-1377, December 2005          
ISSN 1684–5315 © 2005 Academic Journals

Full Length Research Paper

Detection of gfp expression from gfp-labelled bacteria spot inoculated onto sugarcane tissues

Joyce Prisca NJOLOMA¹*, Moriya OOTA², Yuichi SAEKI¹ and Shoichiro AKAO¹

¹Department of Biochemistry and Applied Bioscience, Faculty of Agriculture, University of Miyazaki, Miyazaki, 889-2192 Japan.

²Okinawa Prefecture Agricultural Experimental station, Nishizato, Hirara. 906-0012, Japan.

*Corresponding author. E-mail: jnjoloma@yahoo.com, Tel: +81-985-58-72, Fax: +81-985-58-7206.

Accepted 27 October, 2005

Green fluorescent protein (GFP) as a marker gene has facilitated biological research in plant-microbe interactions. However, there is one major limiting factor in the detection of GFP in living organisms whose cells emit background autofluorescence. In this study, Herbaspirillum sp. B501gfp1 bacterial cells were spot inoculated onto 5 month-old sterile micro-propagated sugarcane tissues to detect if the GFP fluorescence expression could be distinguished from the tissue’s background fluorescence. Stem tissues and leaf sections mounted on glass slides were directly inoculated with a single touch using the tip of a syringe previously dipped into the inoculum containing 10⁸ bacterial cells/ml. We observed that GFP fluorescence could be easily distinguished in the stem than in the leaf tissues. However, the brightness level of the fluorescence varied with time as a result of fluctuations in the bacterial cell density.  The presence of chloroplasts in the leaf tissues of sugarcane requires the use of bright GFP variants when monitoring bacteria-plant interactions using GFP labelled bacteria.

Key words: GFP fluorescence, autofluorescence, bacterial cell density, sugarcane plant tissue.