Germination and Growth on Agar

1. Surface sterilized seeds with 70% ethanol for min, and rinse 3 times thoroughly with deionized water. Then surface sterilize with bleach for 5 min, and rinse with deionized water.
2. Sow seeds on 1.0% agar plus 0.5 X Murashige and Skoog (MS) basal salt medium in Petri dishes.
3. Cold treat at 4^o C for a 3-day interval
4. Place seeds on light cart under white light-treatment (150 m mol m^-2 s^-1). For etiolated (dark-grown seedlings) expose for 1 hour white, then move plates to dark incubator at 23 to 24^o C. 
5. Position plates upright.
6. Photograph seedlings at intervals using the Pixera system (S117).  Label seedling structures.

Histochemical GUS analysis

1. Immerse tissue in the GUS reaction buffer (2 mM X-Gluc, 0.1 mM potassium ferricyanide, 0.1 mM potassium ferrocyanide, 1 mM EDTA, and 50 mM NaPO₄, pH 7.0).
2. Vacuum infiltrate briefly. 
3. Incubated at 37°C for 4 to 16 h.
4. After incubation, clear seedlings methanol or 70% (v/v) ethanol to remove chlorophyll for better visualization.

X-Gluc Stock Solution:  5-bromo-4-chloro-3-indolyl-beta-D-glucuronic acid, cyclohexylammonium salt -D GlcUA [X-Gluc]: cyclohexylammonium salt B6650 10 mg $52.80; 25 mg $79.20). A 10 mg table dissolved in 100 m L [w/v] dimethylformamide = 80 mg/mL (125 mL final vol; 153 mM)

X-Gluc Working Solution:  Add 6.5 uL X-Gluc stock to 500 uL buffer

Buffer:

1 mM EDTA