Add 1 ml of modified Barfoed's reagent.
Groups of 15, 30 and 50 in vitro matured oocytes were transferred to 2, 1 and 0.1 ml of modified Tris-buffered mTBMum (mTBM) and inseminated with frozen-thawed spermatozoa (2000 spermatozoa/oocyte) in a 3×3 factorial experiment.
Experiments were carried out in batch mode using 120-mL serum vials containing 100 mL of modified Postgate growth medium with pH 7 and were sealed with aluminum crimps and butyl rubber stoppers.
Seven species of fungi (Table 1) were maintained as stocks in 100 x 15 mm petri plates containing 15 mL of modified Kirk's medium (Kirk and Fenn [1982]) with 3% (w:v) malt agar at 4°C and pH 5.0.
The pre-cultures (100 μl) were added to 100 ml flasks containing 20 ml of modified fungal minimum medium (FMM) (0.3% yeast extract, 0.2% NaNO3, 0.06% KH2PO4, 0.06% MgSO4·7H2O) with 8% xylose or beechwood xylan.
In this test, we added 0, 0.4, 0.6, 0.8, 0.8, and 1.0 g/L of Tyr to 500 mL flasks containing 200 mL of modified Czapek Dox liquid medium.
The α-L-arabinofuranosidase production was carried out in 250-ml Erlenmeyer flasks containing 5 g of washed dried agro residues (wheat bran, wheat straw, rice bran, rice straw, corn cobs, and maize bran) moistened with 10 ml of modified Mandels-Weber medium containing the following (g/l): urea, 0.3; ammonium sulfate, 1.4; KH2PO4, 0.3; CaCl2, 0.3; MgSO4.7H2O, 0.3; proteose peptone.
In short: the C4 strain was grown in 50 ml of modified Schaeffer's medium (Leighton and Doi 1971) containing Beef extract, 3 g/l; Bactopeptone, 5 g/l; KCl, 2 g/l; Yeast extract, 2 g/l; pH 7, and supplemented with 2 mM MgSO4.7H2O; 1 mM CaCl2; 0.1 mM MnCl2; 1 mM FeSO4; and 0.1%% (w/v) glucose.
Then, 1 mL of the seed culture was transferred into a baffled 500 mL shaking flask containing 100 mL of modified TSB medium with 30 g/L glucose (Nacalai, Kyoto, Japan) as a carbon source, 15 g/L tryptone (Nacalai, Kyoto, Japan) as a nitrogen source, and 5 μg/mL of thiostrepton.
For H+ and Ca2+ measurements, rice roots were grown in hydroponic conditions and placed in plastic Petri dishes (140 × 140 mm) filled-up with 30 mL of modified Clark solution at ¼ strength supplemented with 0 mM or 3.5 mM C HA, excepted for Ca2+ measurements when 100 μM Ca2+ was used.
Shake cultivation was performed according to the following procedure: four 500 mL Erlenmeyer flasks each containing 200 mL of modified Czapek Dox liquid medium (10.0 g/L glucose, 3.0 g/L NaNO3, 1.0 g/L K2HPO4, 0.5 g/L MgSO4, 0.5 g/L KCl, and 0.01 g/L FeSO4) were inoculated with a 0.5 mL aliquot of spore suspension (3.7 × 107 spores/mL).
