Histochemical GUS analysis in transgenic rice

Bio-Protocol Exchange

Concise Method

Switchgrass (Panicum virgatum L.) Promoters for Green Tissue-Specific Expression of the MYB4 Transcription Factor for Reduced-recalcitrance Transgenic Switchgrass

Wusheng Liu^1,2†, Mitra Mazarei^1,6†, Rongjian Ye^1†, Yanhui Peng¹, Yuanhua Shao¹, Holly L. Baxter^1,6, Robert W. Sykes^3,6, Geoffrey B. Turner^3,6, Mark F. Davis^3,6, Zeng‑Yu Wang^4,6, Richard A. Dixon^5,6 and C. Neal Stewart Jr.^1,6*

1. Department of Plant Sciences, University of Tennessee, Knoxville, TN, USA.
2. Department of Horticultural Science, North Carolina State University, Raleigh, NC, USA. 
3. National Renewable Energy Laboratory, Golden, CO, USA. 
4. Noble Research Institute, Ardmore, OK, USA. 
5. BioDiscovery Institute and Department of Biological Sciences, University of North Texas, Denton, TX, USA. 
6. BioEnergy Science Center (BESC), Oak Ridge National Laboratory, Oak Ridge, TN, USA.

Histochemical GUS staining solution

This protocol is detailed description of a method stated in a research article published in Biotechnology for Biofuels 11, 122 (2018). https://doi.org/10.1186/s13068-018-1119-7.

Materials

• X-Gluc (5-bromo-4-chloro-3-indolyl glucuronide cyclohexylammonium salt (Gold Biotechnology, St Louis, MO, USA. Catalog # G1281C2).
• KH₂PO₄ (potassium phosphate monobasic)
• K₂HPO₄ (potassium phosphate dibasic) 
• Triton X-100
• DMSO (dimethyl sulfoxide)
• Distilled water 
• Beaker or flask
• Magnetic stirrer and stir bar
• pH meter
• Graduated cylinder 

Preparation of potassium phosphate buffer at pH 7

• First prepare two separate stock solutions of 1M KH₂PO₄ (acid form) and 1M K₂HPO₄ (basic form). 
• To make 1M KH₂PO₄ (MW= 136.09), dissolve 136.09 g in distilled water to a final volume of 1000 ml.
• To make 1M K₂HPO₄ (MW= 174.18), dissolve 174.18 g in distilled water to a final volume of 1000 ml.
• To create a 1M potassium phosphate buffer at pH 7, mix 61.5 ml of 1M K₂HPO₄ and 38.5 ml of 1M KH₂PO₄. The pH of the obtained solution (100 ml) will be around 7.
• To adjust pH (if necessary), use a pH meter to check the final pH and adjust it by adding more 1M K₂HPO₄ to raise the pH, or KH₂PO₄ to lower it.

Preparation of GUS staining solution

Make fresh immediately prior to use.

• Dissolve 50 mg X-Gluc in 5 ml DMSO
•  Add 10 ml of 1M potassium phosphate buffer at pH 7
• Add two-drop of Triton X-100
• Dilute with distilled water to a final volume of 50 ml

Final concentration in 50 ml staining solution:

• X-Gluc = 1 mg/ml
• Potassium phosphate buffer at pH 7 = 200 mM
• DMSO = 10%
• Triton X-100 = 0.1%