56. Improved histochemical staining for beta-D-glucuronidase activity in monocotyledonous plants

Saskia RUEB and Lambert A. M. HENSGENS

Plant Molecular Biology Research Group MOLBAS, Leiden University, Leiden, The Netherlands

The histochemical detection of beta-D-glucuronidase activity, as decribed by Jefferson is excellent for many dicotyledons. We encountered, however, difficulties with monocotyledons (especially rice). This was mainly due to a poor penetration of the substrate in the tissues. We therefore have examined a variety of fixation conditions for tissues as roots, stem sections, leaves, callus and cell suspensions. Even low concentrations of the mild fixative formaldehyde resulted in a dramatic loss of Beta-D-glucuronidase activity. To avoid this loss of activity we have improved substrate penetration in the tissue without the use of any fixative.

The most consistent results were obtained by partial degradation of cuticle and cell wall components by prolonged incubation with a detergent, followed by vacuum infiltration of the substrate.

Solutions: 
1) 0.1 M phosphate-buffer
  6.66 g/l NaH\2\PO\4\.H2O
  17.66 g/l Na\2\HPO\4\-12H20 (pH 6.8)
2) 0.07% (v/v) Liqui-Nox in 0. 1 M phosphate-buffer, Liqui-Nox is a commercial soap, with a neutral pH, available from ALCONOX, Inc. New York.

3) 0.5 mM K\3\Fe(CN)/6
   2.0 mM 5-bromo-4-chloro-3-indolyl glucuronide (X-Gluc)
  10   mM Na\2\EDTA
   0.1  M phosphate buffer, (pH 6.8)
Staining Procedure: a) Incubate freshly cut or frozen sections or tissues in buffer 2 at 37 deg.C for one hour.

b) Wash two times with 0.1 M phosphate buffer (solution 1).

c) Remove phosphate buffer and add the assay buffer (solution 3).

d) The substrate X-Gluc is infiltrated into the tissue by applying a partial vacuum for 3-4 minutes.

e) Incubate for two hours at 37 deg.C.

f) Staining is intensified by leaving the samples at room temperature for 24 hours.

Whole tissues, callus and suspension culture cells can be stained in this manner. After staining, tissue sections can be cleared with 70% acetone or 5% hypochloride solution to improve contrast.

This work is supported by the Rockefeller Foundation.

References

Jefferson, R. A., 1987. Assaying chimeric genes in plants: The Gus gene fusion system. Plant Molecular Biology Reporter 5(4): 387-405.

____, T. A. Kavanagh and M. W. Bevan, 1987. GUS fusions: Beta-glucuronidase as a sensitive and versatile gene fusion marker in higher plants. EMBO J. 6: 3901-3907.

Hinchee, M. A. W., D. V. Connor-Ward, C. A. Newell, R. F. McDonnell, S. J. Sato, C. S. Gasser, D. A. Fischhoff, D. B. Re, R. T. Fraley and R. B. Borsch, 1988. Production of transgenic soybean plants using Agrobacterium-mediated DNA transfer. Biotechnology 6: 915-922.