Agriculture Reference
In-Depth Information
1.5.5 Bioassay of Plant Growth-Promoting Activities of PS
Bacteria
1.5.5.1 Screening for 1-Aminocyclopropane-1-Carboxylate (ACC)
Deaminase Activity
Using the spot inoculation method, 5
l of each isolated PS bacterium is placed on a
section of plate (marked in 16 equal parts) containing DF (Dworkin and Foster
1958 ) salt minimal medium [g/l: KH 2 PO 4 4; Na 2 HPO 4 6, MgSO 4 ·7H 2 O 0.2,
glucose 2.0, gluconic acid 2.0; citric acid 2.0; trace elements, 1 mg FeSO 4 ·7H 2 O,
10
μ
μ
gH 3 BO 3 , 11.19
μ
g MnSO 4 ·H 2 O, 124.6
μ
gZnSO 4 ·7H 2 O, 78.22
μ
g
μ
CuSO 4 ·5H 2 O, 10
g MoO 3 , pH 7.2 and 2.0 g (NH 4 ) 2 SO 4 as nitrogen source]
supplemented with three mM ACC instead of [(NH 4 ) 2 SO 4 )] and incubated at
28
2 C for 72 h. The bacterial growth should be checked daily as suggested by
Penrose and Glick ( 2003 ). At least one ACC deaminase-positive bacterial strain
should be used as a control in this type of study (Nascimento et al. 2011 ), and all the
samples should be tested in duplicate, and experiments must be repeated at least
three times to ensure the reproducibility of the results.
Quantitative Assay of ACC Deaminase Activity
The ACC deaminase activity of P solubilizers (Ahmad et al. 2013 ) can be assayed
following the method of Honma and Shimomura ( 1978 ) later modified by Penrose
and Glick ( 2003 ). According to this method, the amount of
-ketobutyrate is
measured which is produced by reaction of the enzyme ACC deaminase which
cleaves ACC to
α
-ketobutyrate
produced by this reaction is determined by comparing the absorbance at 540 nm of a
sample to a standard curve of
α
-ketobutyrate and NH 3 . The number of mmol of
α
α
-ketobutyrate ranging between 0.1 and 1 mmol. A
stock solution of 100 mM
-ketobutyrate (Sigma-Aldrich) is prepared in 0.1 M
Tris-HCl, pH 8.5, and stored at 4 C. Just prior to use, the stock solution is diluted
with the same buffer to make a 10 mM solution from which a standard concen-
tration curve is generated. Each in a series of known
α
-ketobutyrate concentrations
is prepared in a volume of 200 ml, 300 ml of the 2,4-dinitrophenylhydrazine reagent
(0.2 % 2,4-dinitrophenylhydrazine in 2 M HCl) (Sigma-Aldrich) is added and the
contents are vortexed and incubated at 30 C for 30 min during which time the
α
α
-ketobutyrate is derivatized as a phenylhydrazone. The colour of the phenyl-
hydrazone is developed by the addition of two ml 2 M NaOH; after mixing, the
absorbance of the mixture is measured at 540 nm. Using this method, the ACC
deaminase activity can be measured in bacterial extracts prepared in the following
manner. The ACC deaminase-positive bacterial strains, for example, P solubilizers
(10 8 cells/ml) are inoculated in Luria-Bertani broth (g/l: tryptone 10; yeast extract
5; NaCl 10; pH 7.5) and incubated in a shaking incubator at 200 rpm for 24-48 h at
28
2 C. Then, cultures are centrifuged at 8,000
g for 10 min at 4 C, and the
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