Methylene blue reduction assay

The method described by Lambert et al. (1985) and

Zhang (2006) was used for the methylene blue reduction

assay. All nodules were thoroughly washed in water to

remove soil particles and excess plant matter. The number

and distribution of root nodules varied from plant to

plant, but were generally no less than ten per legume.

Washed nodules were then placed in small labeled seed

germination plates with filter paper that had been soaked

in a methylene blue reduction solution of 200 mmol/L

iodoacetic acid, 200 mmol/L malonic acid, 2.5 mmol/L

MgCl2, 50 mmol/L K2HPO4, 10 mmol/L Methylene Blue

dye, and adjusted to pH 5.6 with KOH. The nodules were

distributed evenly on the plates, crushed and left to incubate in air for 15 min. The plates were then placed under

a 100% H2 atmosphere in a sealed gas chamber overnight

at room temperature. Plates were removed and photographed using a Canon PowerShot S21S digital camera.

The presence of colorless zones around each nodule represented uptake hydrogenase activity (Hup+) while the

absence of colorless zones represented Hup microsymbionts.

thank u, mr. rostami rad