as an
internal control to reduce variability in all our particle bombardment
experiments (Figure 1A). The expression results (Figure 2B) are reported
as the ratio of luciferase to GUS activity normalized to 100% for BKm3
expression in abscission zones. Expression of luciferase from the test
promoter, the BKm3 construct (Figure 1A), relative to GUS expression from
D35S (i.e., luciferase to GUS ratio) in stems and petioles is approximately
10% of the expression in abscission zones (Figure 2B). This pattern of
expression is very similar to that for native cellulase activity measured
in the same extracts collected from particle bombarded stems, petioles
and leaf abscission zones (LAZ) (Figure 2A).
The VersaFluor fluorometer is inexpensive and easy to use. It has the
necessary sensitivity to reproducibly measure transient GUS activity in
particle gun bombarded plant tissues. In addition, using the Hoechst 33258
dye to quantitate DNA by the VersaFluor fluorometer is reliable, simple,
and accurate.
References
1. Jefferson, R. A., Kavanagh, T. A. and Bevan, M. W., EMBO 6, 3901-3907
(1987).
2. Kay, R., Chan, A., Daly, M. and McPherson, J., Science, 236, 1299-1302
(1987).
3. Koehler, S. M., Matters, G. L., Nath, P., Kemmerer, E. C. and Tucker,
M. L., Plant Molec. Biol., 31, 595-606 (1996).
4. Ow, D. W., Wood, K. V., DeLuca, M., DeWet, J. R., Helinski, D. R.,
and Howell, S. H., Science, 234, 856-859 (1986).
Triton is a trademark of Rohm and Haas. Biolistic is a trademark of E.I.
Du Pont de Nemours and Co. Biolistic technology is exclusively licensed to
Bio-Rad Laboratories.
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