BT 3 optimization of medium for indole 3 acetic acid production using pantoea agglomerans strain PVM

bài báo về sản xuất acid amin

O R I G I N A L A R T I C L E

Optimization of medium for indole-3-acetic acid production using Pantoea agglomerans strain PVM

O.A Apine and J.P Jadhav

Department of Biotechnology Shivaji University, Kolhapur, India

Introduction

Plant growth–promoting rhizobacteria are free living,

which enhances the growth of the plant either directly or

indirectly The mechanism involves nitrogen fixation,

phosphorous solubilization and production of various

phytohormones (Karnwal 2009) The auxins are the

group of phytohormones having indole ring compounds

(Khamna et al 2010) and which play a key role in

stimu-lation of cell division and cell elongation It also controls

the lateral and adventitious root formation and mediates

the tropic response to gravity and light The lower

concentrations of auxin stimulate root elongation,

whereas higher concentration inhibits the root elongation

(Madhaiyan et al 2007)

Diverse groups of micro-organisms, including soil,

epi-phytic and endoepi-phytic bacteria and some cyanobacteria,

were found to synthesize indole-3-acetic acid (IAA) in the

presence of l-tryptophan (Pedraza et al 2004)

Micro-organisms from rhizospheres of various plants synthesize and release auxin as secondary metabolites because of rich substrates exuded from the roots in rhizosphere compared with nonrhizospheric soils (Ahmad et al 2005) The release of l-tryptophan in root exudates may result in its conversion into IAA by rhizosphere microbes (Kravchenko

et al 2004) Several of these groups are implicated in the plant pathogenesis, while others stimulate plant growth (Pedraza et al 2004) The earlier study showed that plant growth–promoting bacteria from different genera (Azospirillum, Enterobacter, Azotobacter, Klebsiella, Alcali-genes faecalis), actinomycetes (Streptomyces olivaceoviridis, Streptomyces rimosus) and fungi (Colletotrichum gloeospo-rioides, Ustilago maydis) have shown to enhance plant growth by the synthesis of IAA (Torres-Rubio et al 2000; Reinekei et al 2008; Khamna et al 2010) The Streptomy-ces griseoviridis K61 and StreptomyStreptomy-ces lydicus WYEC108 were used commercially for IAA production under the trade name Mycostop (Khamna et al 2010)

Keywords

HPTLC, IAA, l-tryptophan, meat extract,

Pantoea agglomerans strain PVM.

Correspondence

Jyoti P Jadhav, Department of Biotechnology,

Shivaji University, Vidyanagar, Kolhapur

416004, India.

E-mail: jpjbiochem@gmail.com

2010 ⁄ 1969: received 2 November 2010,

revised 3 February 2011 and accepted 10

February 2011

doi:10.1111/j.1365-2672.2011.04976.x

Abstract Aims: To optimize the medium components for the production of indole-3-acetic acid (IAA) by isolated bacterium Pantoea agglomerans strain PVM Methods and Results: Present study deals with the production of an essential plant hormone IAA by a bacterial isolate P agglomerans strain PVM identified

by 16S rRNA gene sequence analysis The medium containing 8 g l)1of meat extract and 1 g l)1 of l-tryptophan (precursor) at optimum pH 7, 30C and 48-h incubation gave the maximum production of IAA (2Æ191 g l)1) Effect of IAA synthesized on in vitro root induction in Nicotiana tobacum (leaf) explants was compared with that of control IAA was characterized by high-performance thin-layer chromatography, high-performance liquid chromatography and gas chromatography–mass spectroscopy

Conclusions: Pantoea agglomerans strain PVM was a good candidate for the inexpensive and utmost production of IAA in short period, as it requires sim-ple medium (meat extract and l-tryptophan)

Significance and Impact of the Study: The present report first time showed the rapid, cost-effective and maximum production of IAA No reports are available

on the optimization of particular medium components for the production of IAA This study demonstrates a novel approach for in vitro root induction in

N tobacum (leaf) explants

Pantoea agglomerans is wide spread in many diverse

natural and agricultural habitats, in particular it is

associ-ated with many plants as common epiphytes and

endo-phytes Currently, the genus Pantoea includes seven

species and two subspecies, the majority of which are

associated with plant growth promotion (Barash and

Manulis-Sasson 2009; Sergeeva et al 2007)

Besides extensive research on IAA production by

microbial species, there is still a need to do further

research in this area for process improvement Here, we

report the highest IAA production under optimized

med-ium conditions using a bacterial isolate P agglomerans

strain PVM compared to earlier reports

Materials and methods

Chemicals

l-Tryptophan, meat extract, perchloric acid,

ortho-phos-phoric acid, nitric acid and ethanol were obtained from the

Himedia Laboratories, Mumbai, India All chemicals used

were of the highest purity available and of analytical grade

Screening, isolation and identification of micro-organism

Isolation of the microbial strain capable of IAA

produc-tion carried out from the agricultural soil by enrichment

culture technique The minimal salt medium

supple-mented with l-tryptophan was used for the isolation The

250-ml conical flasks containing the above-stated medium

(100 ml) were prepared in duplicate and inoculated with

1 g of soil sample, followed by incubation at 30C under

shaking conditions at 120 rev min)l for 48 h A loopful

of sample from soil-free enrichments was streaked on

agar plate having media composition as stated earlier

Morphologically distinct colonies were selected for

screening of its IAA-producing ability The most efficient

bacterial isolate is selected and used for further studies

Identification of the isolate as P agglomerans strain PVM

was done by 16S rRNA gene sequence analysis at

Chro-mous biotech Pvt Ltd, Bangalore, India, and the sequence

is deposited in the Gene Bank

Phylogenic analysis

The partial nucleotide sequence of P agglomerans strain

PVM was obtained from Chromous biotech Pvt Ltd

Blasted by using the NCBI server (http://ncbi.nlm.nih.gov/

Blast.cgi), the homologous species were used for phylogenic

analysis The evolutionary history was inferred using the

neighbour-joining method (Saitou and Nei 1987) The

optimal tree with the sum of branch length = 36Æ65209254

is shown The percentage of replicate trees in which the

associated taxa clustered together in the bootstrap test (500 replicates) is shown next to the branches (Felsenstein 1985) The phylogenetic tree was linearized assuming equal evolu-tionary rates in all lineages (Takezaki et al 2004) The clock calibration to convert distance to time was 1 (time per node height) The tree was drawn to scale, with branch lengths in the same units as those of the evolutionary dis-tances used to infer the phylogenetic tree The evolutionary distances were computed using the maximum composite likelihood method (Tamura et al 2004) and are in the units of the number of base substitutions per site Codon positions included were 1st + 2nd + 3rd + noncoding All positions containing gaps and missing data were eliminated from the dataset (complete deletion option) There are total 1393 positions in the final dataset A phylogenetic analysis was conducted in mega4 (Tamura et al 2004)

Organism and culture conditions Bacterial strain (stock culture) was maintained routinely

on nutrient agar containing the following (g l)1): peptone (10), yeast extract (3), NaCl (5), agar (20) and l-trypto-phan (1), stored at 4C until used All the experiments were carried out using above-stated medium The experi-ments were carried out in triplicates at pH 6Æ8, 30C and

at 120 rev min)l unless otherwise stated

pH and temperature optima

To enhance the reproducibility of results and to optimize the biosynthesis process as a whole, the factorial design of experiments, the ‘one factor at a time’ method, was employed in this study Here, the experimental factors are varied one at a time with the remaining factors held constant The pH optima for the production of IAA was determined by varying pH to 3, 4, 5, 6, 7, 8, 9 and 10 Similar experiments were performed to assess the effect of temperature by incubating flasks at 10, 20,

30, 40 and 50C Finally, the produced IAA and residual

l-tryptophan were assayed

Effect of precursor (L-tryptophan) on IAA production The effect of l-tryptophan concentrations on IAA production was studied using medium supplemented with

l-tryptophan at concentrations of 1, 2, 3, 4, 5, 6, 7, 8, 9,

10, 11, 12, 13, 14 and 15 g l)1 followed by incubation at optimum pH and temperature

Consequence of media components on IAA production Effects of various media components on the production

of IAA were assessed by altering the medium composition

accordingly NaCl (2, 4, 6, 8, 10 g l)1), carbon sources

(sucrose, glucose, maltose, lactose, fructose and starch)

and amino acids (l-glycine, l-alanine, l-valine, l-leucine,

l-isoluceine, l-phenylalanine, l-tyrosine, l-tryptophan,

l-aspartic acid, l-glutamic acid, l-histidine, l-arginine,

l-asparagine, l-cystein, l-methionine, l-threonine,

l-pro-line, l-serine, l-glutamine and l-lysine) were used at

(1 g l)1), while nitrogen sources (meat extract, beef

extract, tryptone, yeast extract, peptone) at (10 g l)1)

concentration was investigated

The medium components giving the maximum yield in

minimum time course was preferred for further

optimiza-tion by eliminating the other medium components of

nutrient agar

Effect of incubation time

The effect of incubation time on IAA production was

assayed by incubating bacterial cultures under optimum

conditions up to 168 h Production of IAA and residual

l-tryptophan was measured at every after 24 h

IAA andL-tryptophan assay

The IAA produced was assayed by previously quoted

method (Gordon and Weber 1951) The supernatant

(1 ml) was mixed with two drops of ortho-phosphoric

acid and 4 ml of Salkowski’s reagent followed by

incuba-tion for 30 min in dark, at room temperature The

devel-oped pink colour was measured by recording the

absorbance at 530 nm using UV–Vis spectrophotometer

(Hitachi UV 2800, Tokyo, Japan) Quantification of IAA

was performed using standard curve

l-Tryptophan utilized was determined by estimating

the residual l-tryptophan remaining in the broth by

pre-viously quoted spectrophotometric test (Hassan 1975)

Briefly, 1 ml of cell-free aliquots was taken from the

broth and evaporated on a boiling water bath to dryness,

followed by addition of 1 ml of nitric acid (16 mol 1)l)

and incubation at 50C for 15 min The contents were

then cooled at room temperature following the addition

of 4 ml of sodium hydroxide (5 mol l)l) solution; ethyl

alcohol was used to make the final volume of 10 ml After

mixing the contents, absorbance was recorded at 360 nm

Quantification of l-tryptophan was performed using

stan-dard curve

Tryptophan aminotransferase assay

Preparation of cell-free extract

The bacteria grown in optimized medium at 30C and

120 rev min)l were harvested during exponential phase

after 12 h (centrifuged at 9000 g for 10 min at 4C)

The cell pellets resuspended in the 0Æ5 m borate buffer (pH 8Æ5), and the cells were disrupted by sonication with stroke of 40 Hz for 30 s, total seven strokes keeping 2 min of time interval after each stroke After sonication, the homogenate was centrifuged at 14 000 g for 10 min, and the supernatant was used as a crude enzyme source

Assay The tryptophan aminotransferase activity was determined

by the previously described method (Matheron and Moore 1973; Khandaswami and Vaidyanathan 1973) with some modifications The final assay concentration contained 0Æ5 ml of enzyme extract and 2Æ5 ml of 0Æ5 mol borate buffer (pH 8Æ5) containing 0Æ1 lmol of pyridoxal phosphate, 40 lmol of l-tryptophan and 20 lmol of a-ketoglutarate The reaction mixture was incubated for

30 min at 37C, and the reaction mixture without a-keto-glutarate was used as a control The product formed after enzymatic reaction gave absorbance at 310 nm, so DA310

was monitored before and after the incubation Protein content was determined by the Lowry method (Lowry

et al 1951)

In vitro root induction

In vitro experiment was designed to study the effect of IAA synthesized by P agglomerans strain PVM on root induction in Nicotiana tobacum The three different media combinations were the following: Murashige and Skoog (MS) basal medium as negative control; MS + synthetic IAA (2 mg l)1) as positive control; and MS + IAA synthe-sized by P agglomerans strain PVM (cell-free broth) as test All medium combinations were supplemented with sucrose 3% as per the requirement of the experiment The medium was solidified with 0Æ2% clarigel (Hi-media, India) The pH of the medium was adjusted to 5Æ8 ± 0Æ05 before autoclaving Surface-sterilized explants were placed

in test tubes (25 · 100 mm), containing 15 ml of med-ium, and culture bottles (200 ml capacity), containing

40 ml of medium, and were autoclaved at 15 psi and 121C for 20 min All cultures were maintained at

25 ± 2C with 16 h light and 8 h in the dark

Analytical studies

High-performance thin-layer chromatography (HPTLC) The analysis of produced IAA was performed by using HPTLC system (CAMAG, Muttenz ⁄ Switzerland) Station-ary-phase HPTLC silica gel 60 F254 (Merck, Germany), predeveloped with methanol, was dried at 120C for

20 min, cooled to room temperature and equilibrated

with the relative humidity of the laboratory The

sample was applied on to the plate by spray-on technique

(nitrogen as spray gas) Two microlitres of standard IAA

(2 mg ml)l), and the broth before and after incubation

(IAA produced) were loaded, by using TLC

sample-loading instrument (CAMAG LINOMAT 5) The plate

was developed by using saturated twin-trough chamber;

10 ml of developing solvent n-hexane ⁄ ethyl acetate (4 : 6)

in front trough of 20 · 10 cm chamber The chamber

was saturated for 20 min prior to plate development

Developing distance is 60 mm from the lower edge of the

plate After development, the developed plate was scanned

in the absorbance mode with slit dimension of 5 · 0Æ45

mm, scanning distance 5–65 mm, at 280 nm, using

deuterium lamp by using TLC scanner The results were

analysed using HPTLC Win cats planar chromatography

manager, ver 1.4.4.6337 software (CAMAG Muttenz ⁄

Switzerland)

High-performance liquid chromatography (HPLC)

HPLC analysis was carried out (Waters model no 2690;

Waters Corp., Milford, MA, USA) on C18 column

(sym-metry, 4Æ6 mm·250 mm) by using HPLC-grade methanol

as mobile phase (75 : 25) with flow rate of 1 ml min)1

for 10 min and UV detector at 280 nm The standard

IAA and sample IAA produced in broth were prepared in

HPLC-grade water and used for further analysis

Gas chromatography–mass spectroscopy (GC–MS)

Extraction of sample

The broth (incubated for 48 h) was centrifuged at 6600 g

for 15 min, and the supernatant was collected The

super-natant was extracted with double volume of ethyl acetate

in separating funnel The ethyl acetate fraction was

recov-ered into a new flask and evaporated The residue was

dissolved in methanol and used for analysis

GC–MS analysis

A GC–MS analysis of sample was carried out using a

Shimadzu (Nakagyo-Ku, Kyoto, Japan) 2010 MS Engine,

equipped with integrated gas chromatograph with a

Res-tek column (0Æ25 mm, 60 m; XTI-5) Helium was used as

carrier gas at a flow rate of 1 ml min)1 Injector and

detector temperatures were both set to 280C The oven

temperature was held at 80C, for 2 min, then

pro-grammed to rise from 80 to 200C at 10C min)1 and

then finally programmed from 200 to 280C at

20C min)1 rate for 7 min The compounds were

identi-fied on the basis of mass spectra and by using the NIST

library

Results

Identification and phylogenic analysis The potent IAA-producing bacterial species, which was iso-lated, was identified as P agglomerans strain PVM The phylogenic position of P agglomerans strain PVM in relation

to other species of this genus is illustrated in Fig 1a; the digits adjacent to nodes are the statistical frequency of the indicated species The numbers shown in parentheses are accession numbers of different species The strain is deposited in GenBank under the accession number GU929212

pH and temperature optima

It was observed that P agglomerans strain PVM showed maximum IAA production of 1Æ441 g l)1 utilizing 0Æ752 g l)1 l-tryptophan at pH 7 (Fig 1b) Similar study with temperature optimization showed that 1Æ53 g l)1 IAA was produced by utilizing 0Æ804 g l)1of l-tryptophan (Fig 2a), when incubated at 30C

Optimum l-tryptophan concentration The maximum production of IAA 158% (1Æ577 g l)1) was achieved by utilizing 78% (0Æ783 g l)1) l-tryptophan, when medium was supplemented with 1 g l)1 concentra-tion of l-tryptophan as a precursor (Fig 2b)

Effect of media components The effect of various concentrations of NaCl on IAA production was evaluated The higher amount of IAA production (1Æ176 g l)1) was achieved by utilizing (0Æ780 g l)1) l-tryptophan in NaCl (4 g l)1)-supplemented medium (Fig 2c)

Sucrose was best utilized by the organism and gave 1Æ308 g l)1of IAA with 0Æ738 g l)1utilization of l-trypto-phan (Fig 2d) On the other hand, carbon sources like glucose and fructose gave the moderate IAA production 0Æ985 and 0Æ945 g l)1, respectively

When the effect of various nitrogen sources on IAA production with respect to tryptophan utilization was studied (Fig 3a), it was concluded that meat extract gave higher production of IAA (2Æ104 g l)1) by utilizing (0Æ803 g l)1) l-tryptophan as compared to other nitrogen sources Hence, it was further optimized, which gave (2Æ191 g l)1) IAA utilizing (0Æ826 g l)1) l-tryptophan at

8 g l)1meat extract

Also, when the effect of amino acids on IAA produc-tion was studied in the presence of l-tryptophan (Fig 3c), it was observed that l-tryptophan was solely the

best source for IAA production in comparison with the

combinations with the other amino acids tested

Optimum incubation time

The effect of incubation time was studied under

opti-mized cultural conditions The production of IAA and

utilization of l-tryptophan was directly proportional to

the incubation time; after 48-h incubation IAA

produc-tion enhanced by twofold (2Æ059 g l)1) utilizing

(0Æ865 g l)1) l-tryptophan (Fig 3d)

Tryptophan aminotransferase activity

The crude tryptophan aminotransferase activity was

found to be 312Æ44 U mg)l of enzyme after 12 h of

incu-bation, and thereafter enzyme activity slightly decreased

In vitro root induction Pantoea agglomerans strain PVM showed more induc-tion of roots compared with the leaf explants grown

on the MS medium supplemented with synthetic IAA (positive control) The negative control (MS basal) does not show the induction of roots in the N tobacum leaf explants (Fig 4) This confirms the produced IAA will

be better option for the in vitro root induction in plants

Analysis of IAA HPTLC analysis showed the same peak profile for the broth after incubation and standard IAA (Fig 5e) The Rf value of standard IAA was 0Æ57 However, the broth obtained after incubation showed the Rf 0Æ57 value

0 0·2 0·4 0·6 0·8 1 1·2 1·4 1·6

pH

Enterobacter sp AN2 (GQ451698·1) Enterobacter sp CO 8-9 (EU181139·1) Enterobacter sp rif200834 (FJ527677·1) Enterobacter sp E4M-U (GQ478275·1) Enterobacter sp KK1 (GQ871449·1) Enterobacter sp pp9c (GQ360072·1)

Enterobacter sp WAB1938 (AM184277·1) Acinetobacter radioresistens strain Philippines-11 (EF446895·1)

Pantoea sp P102 strain P102 (AF394539·1)

Enterobacter sp SPi (FJ405368·1) Endophytic bacterium EH68 (GU339293·1)

Enterobacter sp DHM-1T (FJ745300·1) Enterobacter sp R4M-Q (GQ478271·1) Pantoea agglomerans strain P29 (DQ356903·1)

Pantoea agglomerans strain PVM (GU929212·1)

Proteobacterium symbiont of Nilaparvata lugens clone TM58 (FJ774962·1) Enterobacter cloacae strain IHB B 1374 (GU186117·1)

Pantoea sp M3 (EF192586·1) Enterobacteriaceae bacterium GIST-WP2s1 (EF428984·1)

20 0

0

3 0 8

0

1

1

5

(a)

(b)

Figure 1 (a) Phylogenic tree of the Pantoea

agglomerans strain PVM and related

organ-isms were aligned based on 16S rRNA

sequences (neighbour-joining tree) Scale

bar – number of nucleotide changes per

sequence position The number at nodes

shows the bootstrap values obtained with

1000 resembling analysis (b) Optimization of

pH for IAA production; IAA produced ( ),

L -tryptophan used ( ) IAA, indole-3-acetic

acid.

similar to that of standard IAA Broth before incubation

do not show the presence of prominent peak

The HPLC elution profile of standard IAA showed

major peak at retention time 2Æ86 min, while elution

profile of the broth after incubation (IAA produced)

confirmed the production of IAA with peak at retention

time 2Æ85 min (Fig 5f)

GC–MS analysis of the extracted samples after

incuba-tion revealed presence of IAA The gas chromatogram

showed presence of a major peak at retention time

11Æ9 min, which was analysed by monitoring the

proton-ated molecular ion of methyl IAA molecular ion m ⁄ z 189

together with the major fragment ion at m ⁄ z 130, and

our results are in good agreement with previously

described GC–MS analysis by Muller et al 2002 (Fig 5g)

Discussion

Present work mainly concerned with the IAA production

potential of P agglomerans strain PVM, optimization of

medium components and in vitro root induction in

N tobacum Pantoea agglomerans strain PVM used in this study produced different levels of IAA in the presence of

l-tryptophan under various physicochemical conditions Acidic and alkaline conditions seemed to hamper the production of IAA as well as growth of organism also, elevated or decreased temperatures from 30C seemed

to affect IAA production Literature survey revealed that Klebsiella strain K8 produces 0Æ0169 g l)1 IAA at pH 8 while 0Æ0179 g l)1 of IAA produced at 37C (Sachdev

et al 2009) The pH does not affect IAA production in Azospirillum brasilense SM It will grow in the tempera-ture range of 25–37C with the optimum being 30C Deviations from the optimum temperature allowed signif-icant improvement in IAA biosynthesis (Malhotra and Srivastava 2009) Industrially important white-rot fungus Lentinus sajor-caju gave 0Æ18 g l)1 of IAA production at

pH 7Æ5 and 30C in dark (Yurekli et al 2003) The IAA production and utilization of l-tryptophan decreased with increasing l-tryptophan concentration The earlier

0

0·2

0·4

0·6

0·8

1

1·2

1·4

1·6

1·8

Temperature (°C)

0 20 40 60 80 100 120 140 160 180

L -tryptophan (g l –1 )

0

0·2

0·4

0·6

0·8

1

1·2

1·4

NaCl (g l –1 )

0 0·2 0·4 0·6 0·8 1 1·2 1·4

10 20 30 40 50 1 2 3 4 5 6 7 8 9 10

Sucrose Glucose Maltose Lactose Fructose Starch

Carbon source (1g l –1 )

(a)

(b)

Figure 2 (a) Optimization of temperature for IAA production; IAA produced ( ), L -tryptophan used ( ) (b) Effect of L -tryptophan on IAA production; IAA produced ( ), L -tryptophan used ( ) (c) Effect of NaCl on IAA production; IAA produced ( ), L -tryptophan used ( ) (d) Effect of carbon sources on IAA production; IAA produced ( ), L -tryptophan used (h) IAA, indole-3-acetic acid.

reported production in Bacillus megaterium MiR-4,

Bacillus sp NpR-1, Bacillus subtilis TpP-1 and Bacillus

licheniformis FiR-1 that produced 0Æ0927, 0Æ0681, 0Æ0654

and 0Æ0626 g l)1, respectively, at 1 or 1Æ2 g l)1 l -trypto-phan (Ali et al 2010) Rhizobium sp isolated from root nodules of Dalbergia lanceolaria and Roystonea regia also

0

0·5

1

1·5

2

2·5

Nitrogen source (1%)

0 0·5 1 1·5 2 2·5

Meat extract (g l–1)

0

0·2

0·4

0·6

0·8

1

1·2

1·4

1·6

-glycine L-alanine L -v

y L-tyrosine

-glutamic L-histidine L-arginine

-methionine L -threonine L -proline L -ser

Amino acids (g l –1 )

0 0·5 1 1·5 2 2·5 3

Meat e

xt.

Beef e

xt.

Tryptone Yeast e

xt.

Peptone

1 2 3 4 5 6 7 8 9 10

24 48 72 96 120 144 168

Time (h)

Figure 3 (a) Effect of nitrogen sources on the IAA production; IAA produced ( ), L -tryptophan used (h) (b) Optimization of meat extract for IAA production; IAA produced ( ), L -tryptophan used ( ) (c) Effect of amino acids on IAA production; IAA produced ( ), L -tryptophan used ( ) (d) Effect of incubation time on IAA production; IAA produced ( ), L -tryptophan used ( ) IAA, indole-3-acetic acid.

Figure 4 In vitro root induction (a) negative control (MS basal medium), (b) positive control (MS basal + synthetic IAA) and (c) test (MS

basa-l + IAA synthesized by Pantoea aggbasa-lomerans strain PVM) MS, Murashige and Skoog; IAA, indobasa-le-3-acetic acid.

produced IAA at 2Æ5 and 3 g l)1 l-tryptophan

concentra-tion, respectively (Basu and Ghosh 2001; Ghosh and Basu

2002) Curtobacterium plantarum 6-I, Streptomyces sp 3s

and Pseudomonas fluorescens 540 produced IAA 0Æ078,

0Æ020 and 0Æ067 g l)1, respectively, at 0Æ4 g l)1l-tryptophan

(Merzaeva and Shirokikh 2010) Pantoea agglomerans

strain PVM reported here gave maximum IAA production

at lower l-tryptophan concentration compared with

ear-lier reports

Abiotic stress like the presence of salt in the medium

hampers the production of IAA Increasing salt stress will

reduce the production of IAA and the utilization of the

precursor molecule Pantoea agglomerans strain PVM

reported here gave relatively highest yield of IAA in the

presence of sucrose as carbon source Earlier studies

regarding Rhizobium strains vary in their utilization and production of IAA with different carbon sources The Rhizobium strains 12, 16 and 18 require sucrose and Rhizobium strain 13 and Rhizobium sp from Cajanus cajan require glucose for maximum production of IAA (Datta and Basu 2000; Shridevi and Konada 2007) However, white-rot fungus Lentinus sajor-caju showed 0Æ18 g l)1 of IAA production in glucose-containing medium, but use of sucrose instead of glucose resulted in a substantial decrease

in IAA biosynthesis (Yurekli et al 2003)

Meat extract gave the maximum IAA production among the various nitrogen source tested Hence, the meat extract was further optimized at 8 g l)1 to give highest IAA production (2Æ191 g l)1) (Fig 3b) In the presence of l-tryptophan, only P agglomerans strain PVM

A a

B

(f)

(g)

900·0

All trade at 254 nm

700·0 500·0 400·0 200·0 100·0 0·0 0·00

2·00

Minutes

1·50 1·00

1·00

2·00

Minutes

1·00

0·50 0·00

0·25 0·20

0·10 0·15

0·05 0·00

1·0

0·5

0·0 50

51 77 103 146 189 130

0·10 0·20

0·70 0·80

0·0 100·0 200·0 400·0 600·0 700·0

900·0 (AU)

(mm)

(Rf) (AU)

Figure 5 (e) Three-dimensional graph generated after scanning of high-performance thin-layer chromatography plate: (a) standard IAA, (b) broth before incubation and (c) broth after incubation (f) HPLC elution profile of (A) standard IAA and (B) HPLC elution profile of broth after incubation (g) Gas chromatography–mass spectroscopy analysis of extracted broth after incubation showed protonated molecular ion of methyl IAA molecu-lar ion m ⁄ z 189 together with the major fragment ion at m ⁄ z 130 IAA, indole-3-acetic acid; HPLC, high-performance liquid chromatography.

gave the maximum production of IAA The earlier study

revealed that Rhizobium strain 16 showed maximum

growth and higher IAA production, when medium

supplemented with l-glutamic acid as nitrogen source

(Shridevi and Konada 2007) Pantoea agglomerans strain

PVM reported here gave the maximum production of

IAA within short incubation period The synthesized IAA

was characterized by various analytical techniques like

HPTLC, HPLC and GC–MS For the first time, in vitro

root induction was observed by using IAA The

synthe-sized IAA shows in vitro root induction in N tobacum;

experimental results showed that IAA-containing broth

shows more root induction than the controls The broth

containing IAA can be effectively used in the field

appli-cation for enhancement of rooting in the agricultural

crops

Pantoea agglomerans strain PVM was a good candidate

for the inexpensive and utmost production of IAA in

short period, as it requires simple medium (meat extract

and l-tryptophan) Further studies regarding scale up of

IAA production and its purification is underway

Acknowledgements

We thank the Department of Biochemistry, Shivaji

University, Kolhapur, and Mr S.N Surwase, Mr U.B Jagtap

and Mr S.S Phugare for valuable technical assistance

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