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Using the VersaFluor Fluorometer to Quantitate GUS Expression in Plant Tissues Bombarded With the Biolistic PDS-1000/He System

0 minutes. The GUS marker enzyme, originally from E. coli, is resistant to heat inactivation at 55 C; however, heating significantly reduces endogenous GUS-like activity in plant extracts. Following incubation, the mixture is combined with 20 l of methanol, which further reduces background GUS-like activity (Kosugi et al., 1990), and 12.5 l of 10 mM MUG. Immediately after addition of MUG, 10 l of the mixture is removed and combined with 2 ml of 0.2 M sodium carbonate to stop GUS conversion of MUG to MU. These tubes are labeled T0. The remaining mixture is incubated at 37 C overnight and the T0 tubes stored in the dark at room temperature. Following incubation at 37 C, 10 l of the mixture is combined with 2 ml of 0.2 M sodium carbonate and the tube labeled T12.

MU standards are prepared at 50, 100 and 500 nM in 0.2 M sodium carbonate. The VersaFluor fluorometer is warmed for 15 minutes before conducting the assay. The instrument has low, medium and high gains. The range of MU standards described above is best measured at a medium gain.

The instrument is zeroed with a blank consisting of 0.2 M sodium carbonate. First, the highest standard, 500 mM, is measured and set to equal 5,000 relative units with the range option of the fluorometer. Next, the two remaining standards are measured to ascertain a linear response. Finally, the fluorescence of extracts in the T0 and T12 tubes is measured and recorded.


Results and Discussion
Fusion of the enhanced CaMV 35S promoter (Kay et al., 1987) to the GUS reporter gene creates a gene construct (D35S) that expresses GUS constitutively in transformed plant tissues. Inclusion of this construct is used
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