Symbiotic Fungi: Principles and Practice (Soil Biology)

192 Z.M. Solaiman
11.2.2 Isolation of Arbuscules and Intraradical
Hyphae without Enzymatic Digestion
11.2.2.1 Materials
Waring blender, microscope, slides, centrifuge machine, tubes.
11.2.2.2 Chemicals
Washing buffer: Prepare by mixing 0.3 M mannitol, 0.01 M Tris-HCl of pH 7.4,
1 mM DTT
11.2.2.3 Protocol
Isolation of arbuscules outlined here according to the method of Senoo et al (2007).
1. Immerse fresh roots of mutant (har1)ofLotus japonicus L. in a cold solution of
0.5 mM CaSO4 for a few minutes, and then cut into 5 mm length. Collect 5g of
the root pieces on nylon mesh (50 mm), and wash with washing buffer.
2. Transfer washed root pieces into a 100 ml homogenizer of Waring blender with
40 ml of cold washing buffer, and homogenize for 30 s at 6,000 rpm.
3. Filter homogenate through two layers of cheesecloth, and again homogenize the
residue with 40 ml of cold washing buffer. Repeat this homogenization/filtration
process two more times.
4. Filter through 50 mm and then 30 mm nylon mesh. Collect residue on the 30 mm
nylon mesh with a Pasteur pipette as the arbuscule and hyphae fractions. Do all
the procedures at 0–4 C if possible.
5. Store precipitate and count under microscope by staining using appropriate
methods.
11.2.3 Measurement of Metabolic Activity of Isolated
Arbuscules and Hyphae
11.2.3.1 Materials
Microscope, slides, needles.
11.2.3.2 Chemicals
0.25 M sodium succinate, 0.5 mM MgCl 2, nitroblue tetrazolium (1 mg ml 1 ), 4 mM
a-naphtyl acid phosphate, Fast Blue RR (0.6 mg ml 1 ), 0.1 M Tris-HCl buffer
(pH 8.5), 0.1 M sodium acetate buffer.