Non‐destructive detection of firefly luciferase (LUC) activity in single plant cells using a cooled, slow‐scan CCD camera and an optimized assay

Abstract

A flexible, comparatively inexpensive system based on a liquid nitrogen‐cooled slow‐scan CCD (charge coupled device) camera is presented, which can be employed for quantitative low‐light (bioluminescence, chemiluminescence or fluorescence) imaging. Using this camera system and the firefly luciferase (LUC) as a screenable marker, transgenic tobacco lines have been produced by direct gene transfer. Bioluminescence emitted from single tobacco cells transiently expressing the firefly luciferase gene (Luc) as well as from stably transformed calli, regenerated shoots, plantlets and T1 seedlings could be monitored in vivo with no effect on the viability of the material analysed. The patterns of light emission from sections through Luc‐expressing leaves and bioluminescent single protoplasts isolated from such leaves were also imaged microscopically. The assay used to detect in vivo LUC activity was optimized by quantifying bioluminescence emitted from Luc‐expressing tobacco protoplasts and leaves incubated in different substrate solutions and determining the kinetics of light emission during incubation in the substrate solution.

From the Concept of Totipotency to Biofortified Cereals

“…He left for a group leader position at the International Rice Research Institute (IRRI) in the Philippines, became a full professor in Calcutta, and is now a deputy director general (crop science) in the Indian Council of Agricultural Research. Gunther Neuhaus optimized microinjection for transformation of precursor cells of somatic embryos (40,41,48,85,86), moved to a full professor position at the University of Freiburg, and is currently the vice rector for research there. German Spangenberg, a fresh PhD when he joined, was so impressive that I promoted him early on to develop his own group, and within a few years he became the world leader in forage grass biotechnology (54, 89-91, 92, 94, 101, 102); since the mid-1990s, he has been a full professor in Melbourne, Australia.…”

Luciferase Gene Expressed in Plants, Not in Agrobacterium

“…Luciferin uptake has been achieved by a variety of methods, including spraying (Millar et al 1992(Millar et al , 1995a, watering (Wood and DeLuca 1987), and petiole feeding (Barnes 1990). Bioluminescence of LUC-expressing plants has been visualized and/or quantified using X-ray film (Ow et al 1986;Wood and DeLuca 1987), cooled, slow-scan CCD cameras (Kost et al 1995), photon-counting cameras and luminometers (Mankin et al 1997). Most lowlight photodocumentation systems can be adapted for this purpose (Hooper et al 1994); however, alight-tight imaging box is critical for keeping light noise to a minimum (Millar et al 1992;Hooper et al 1994).…”

“…The luciferase gene (luc) from the North American firefly Photinus pyralis has been used as a reporter gene in a variety of eukaryotic (Ow et al 1986;Millar et al 1992;Kost et al 1995) and prokaryotic (Wood and DeLuca 1987) organisms. Luciferase assays are facilitated in plants by the lack of endogenous luciferase activity (Abeles 1986).…”

“…Although this review focuses upon the firefly luciferase, the techniques for visualizing bioluminescence and delivering substrate are applicable to other bioluminescence systems such as the Renilla reniformis luciferase system (Mayerhofer et al 1995). Regardless of the system, bioluminescent activity is best detected using a combination of a photon-counting camera (Mankin et al 1997) or cooled, slow-scan CCD camera (Kost et al 1995) for tissue specificity and a luminometer for quantitative analysis (Millar et al 1992;Mankin et al 1997). Luciferase assay quantitation is facilitated by a broad linear range, which can extend over eight orders of magnitude (Wood 1991).…”

High efficiency transient and stable transformation by optimized DNA microinjection intoNicotiana tabacumprotoplasts

“…Immediately after adding the substrate solution the bioluminescence emitted during 10 min was imaged macroscopically through a 50 mm f/1.2 photographic lens using a cooled, slow scan CCD camera as described by Kost et al (1995). The substrate solution was removed after the assay and the cells were washed twice with 200 iA PNT medium.…”