Agriculture Reference
In-Depth Information
biomass of P solubilizers is harvested. The supernatant is removed, and the cells are
washed with 5 ml DF salts minimal medium. Following an additional centrifugation
for 10 min at 8,000
g
at 4
C, the cells are resuspended in 7.5 ml DF salts minimal
medium in a fresh culture tube. Just prior to incubation, the frozen 0.5 M ACC
solution is thawed, and an aliquot of 45 ml is added to the cell suspension to obtain a
final ACC concentration of 3.0 mM. The bacterial cells are re-shaken in the
incubator to induce the activity of ACC deaminase at 200 rpm for 24 h at the
same temperature as is done for overnight-incubated cultures. The bacteria cultures
are harvested by centrifugation at 8,000
g
for 10 min at 4
C. The supernatant is
removed, and the cells are washed by resuspending the cell pellets in 5 ml 0.1 M
Tris-HCl at pH 7. Each bacterial cell pellet, prepared as described above are
suspended in 1 ml of 0.1 M Tris-HCl, pH7.6 and transferred to a 1.5-ml micro-
centrifuge tube. The contents of the 1.5-ml micro-centrifuge tube are spun at
16,000
g
for 5 min, and the supernatant is removed. The pellet is suspended in
600 ml of 0.1 M Tris-HCl, pH 8.5. A 30
l of toluene is added to the cell suspension
and vortexed at the highest setting for 30 s. At this point, a 100-ml aliquot of the
“toluenized cells” is set aside and stored at 4
C for protein assay by Lowery
et al. (
1951
) method at a later time. The remaining toluenized cell suspension is
immediately assayed for ACC deaminase activity. All sample measurements should
be carried out in duplicate. 200
μ
l of the toluenized cells are placed in a fresh 1.5-ml
micro-centrifuge tube; 20 ml of 0.5 M ACC is added to the suspension, briefly
vortexed and then incubated at 30
C for 15 min. Following the addition of 1 ml of
0.56 M HCl, the mixture is vortexed and centrifuged for 5 min at 16,000
μ
g
at
room temperature. One ml of the supernatant is vortexed together with 800 ml of
0.56 M HCl. Thereupon, 300 ml of the 2,4-dinitrophenylhydrazine reagent (0.2 %
2,4-dinitrophenylhydrazine in 2 M HCl) is added to the glass tube; the contents are
vortexed and then incubated at 30
C for 30 min. Following the addition and mixing
of 2 ml of 2 N NaOH, the absorbance of the mixture is measured at 540 nm.
1.5.5.2 Quantitative Assay of Indole Acetic Acid
Indole-3-acetic acid (IAA) synthesized by P solubilizers (Wani and Khan
2010
;
Ahemad and Khan
2012
) is quantitatively evaluated by the method of Gordon and
Weber (
1951
), later modified by Brick et al. (
1991
). For this, the PS bacterial strains
are grown in Luria-Bertani (LB) broth. Luria-Bertani broth (100 ml) having 0, 50,
100, 200, 400 and 500
g/ml tryptophan is then inoculated with 1 ml culture (10
8
cells/ml) of PS cultures and incubated for 3, 6, 9 and 12 days at 28
μ
2
C with
shaking at 125 rpm. After incubation, 5 ml of culture of each treatment is spun
(9,000
lof
orthophosphoric acid and 4 ml of Salkowski' reagent (2 % 0.5 M FeCl
3
in 35 %
perchloric acid) and incubated at 28
g
) for 15 min, and an aliquot of 2-ml supernatant is mixed with 100
μ
2
C in darkness for 1 h. The absorbance of
developed pink colour is read at 530 nm. The IAA concentration in the supernatant
is determined using a calibration curve of pure IAA as a standard. The experiment
should be repeated three times on different time intervals.
