1.      Excise root and leaf segments, wash with 100mM potassium
phosphate buffer (pH7.0) for 3 times.

2.      Immerse in the Gus substrate solution, 37C, dark, 12-24h

3.      Rinse in phosphate buffer

4.      Fix for 4h or longer

5.      Rinse in the same buffer

6.      Observe as whole specimens or as sections.

1M potassium phosphate buffer (pH7.0)

K2HPO4: 34.8g —>200ml

KH2PO4: 27.2g—->200ml

184.5ml K2HPO4+115.5ml KH2PO4—-> 300ml 1M potassium phosphate buffer
(pH7.0)

Gus substrate solution

Volume(500ml)

Final concentration

Potassium ferricyanide

82mg

0.5mM

Potassium ferrocyanide

105.6mg

0.5mM

Potassium phosphate buffer (pH7.0, 1M)

50ml

100mM

Store at -20C. Add 1mM x-gulc (MW521.8~~0.5mg/ml) freshly when use.

Fix solution

2.5% glutaraldehyde

200mM sodium cacodylate, pH7.2