INTRODUCTION:

In recent years, it has been claimed that microbial synthesis of plant growth regulators (PGR) becomes very important for commercial production, which are widely used in the agriculture and horticultural fields. Plant growth hormones are synthesized in minute amounts and influence many physiological processes in plant growth and development. These hormones are not only synthesized by higher plants^22,9, they have also been synthesized by microorganisms ^{27,29,20,6,9,17,24} . IAA is the first hormone identified and having many commercial uses in agriculture and horticultural crops. There is much evidence that bacteria produce IAA in culture media^{14,21,1,12,13}which can be exploited for commercial uses. ABA was the first inhibitory hormone known to be involved in the regulation of plant growth⁴. One of the important roles of ABA is stress management applications. Stress is one of the important challenges to address in the present scenario of food production and to meet the increasing population demand. The synthetic compounds and analogs available are coupled with high cost of production and less efficiency than the natural hormones and are active only in a few assays but not in others². To exploit the economic value of IAA and ABA / hormones to increase food production, a biological method for production of growth regulators in higher concentrations is required. This can be achieved through identifying microorganisms and enhancing the plant growth regulators produced by them.

Since hormones are produced in small quantities, accurate analysis of them is very important. Specific bioassays are available for many hormones but give only qualitative analysis. So need to have technique to detect and quantify the same, one such method is HPLC. Since the first reports on the determination of plant hormones by HPLC in 1970s^25,8,26, the technique has been used with ever increasing frequency in plant hormone studies, the hormones being measured by the methods^3,8, reversed and normal phase HPLC.The present investigation was undertaken to study the growth hormones of MSF34 strain isolated from Nilgiri forest soil. An extraction and purification procedure useful for routine analysis of IAA and ABA was tried and details of this combined determination method was studied by HPLC.

MATERIALS AND METHODS:

In the present paper, we describe an extraction, purification and combined determination method by HPLC useful for routine analysis of IAA and ABA in methylotrophs, The Methylotrophs were isolated and grown in MM medium containing KNO_3, MgSO₄, CaCl_2, Na₂HPO_4, NaH₂PO_4, FeSO_4, CuSO_4, H₃BO_3, MnSO_4, ZnSO₄and MoO₃ in a litre of distilled water and sterilized in an autoclave at 121^oC of 15 lbs pressure for 20 minutes. Sterilized medium was allowed for slight cooling and 5mL of Methanol was added to the medium. The medium was inoculated with the MSF34 under aseptic conditions and incubated in a mechanical shaker. After the incubation of 36 hrs, cultures were centrifuged at 10,000 rpm. Pellets and supernatant were then subjected to extraction of ABA and IAA¹¹shown in figure.1.

Reagents: Methanol (GR grade, redistilled in glass before use). Acetic acid, ethyl acetate and petroleum ether (GR grade, Shanghai chemicals), IAA and ABA (GR grade, Sigma). All solvents were filtered through 0.45µm MF Millipore filters. IAA and ABA were dissolved in methanol (1.0 mg ml^-1) and used as standard samples.

Extraction:

Fig.1: Extraction and purification procedure of IAA and ABA of methylotrophs-MSF34

Qualitative analysis:

Wheat coleoptiles segment elongation bioassay for IAA

Wheat seeds (Triticum aestivum L. Cv. Norin No.10) were surface sterilized in 1% mercuric chloride for 10 min, then washed thoroughly, and soaked in distilled water for four hours. These seeds were sown in plastic trays containing sterile and saturated vermiculate and grown for 96 hrs in dark at 25±2°C. Coleoptiles measuring around 30mm length were selected and decapitated by removing 2-3mm from the apex. Thirty minutes after removal of tip, 10 mm segments were excised just below the top cut end. Ten uniform segments were collected, surface dried and placed in petriplates containing different concentrations of IAA (0.01 to 1000 ppm) MSF34 extract and microbial spent medium with or without dilution, and incubated in dark for 24 hrs. Measurements were obtained by removing the sections from the petriplates. The segments were arranged end to end by lining on a wet glass plate and lengths were measured using a scale.

Cucumber hypocotyls elongation bioassay for ABA

A simple bioassay was carried out to determine the inhibition by abscisic acid (ABA) on cucumber (Cv. Samber) hypocotyl elongation. 10mm segments of 3-day-old dark grown cucumber hypocotyls were excised from immediately below the cotyledon and used for the assay. The excised hypocotyls were floated in petriplates containing different concentrations of ABA ranging from 10^-3 to 10^-9 M, MSF34 extract and microbial spent medium with or without dilution. The petriplates were incubated in dark for 20 h followed by an exposure to light for 24 h. At the end of the incubation period inhibition of hypocotyl segments were measured. Bioassays were done with three replications.

Quantitative analysis by HPLC

Chromatograph conditions

A Waters Assoc. ALC/GPC 401 Liquid Chromatography, with two Module 510 pumps, Module 721 programmer controller, Module U6K injector and Module 441 UV monitors, operating at 254 nm. Inertsil ODS 3V C18 (250x4.5 mm I.D.) column and Z-Module redial compression system was used, the isocratically eluted with MeOH-2% HAc-H₂0 (40:40:20) at a flow-rate of 1.5 ml min^-1. The standard used was ABA in the concentration of 1mg/ml of ethanol (1000 ppm).

RESULTS:

IAA Bioassay:

The influence of different concentrations of synthetic IAA, extract, microbial and spent medium on the elongation of wheat coleoptile segments were studied and results were presented as percentage over control in Table.1. The results indicated that both IAA as well as microbial spent medium were effective in inducing elongation of coleoptile segments but not in cell extracts. The increase in elongation of coleoptiles segments were linear with increase in concentrations of IAA from 0.01 to 10 µM and diluted microbial spent medium from 1:10 to 1:30. However undiluted microbial spent medium and undiluted cell extracts showed less significant growth of coleoptile segments.

Table 1: Influence of different concentrations of IAA, MSF 34 extract and microbial spent medium on elongation of wheat coleoptile segments

Treatments		% Growth over control
IAA(uM)	0.01	3.2
	0.1	21.0
	1.0	38.2
	10	42.6
	100	40.8
	1000	32.9
Microbial spent medium	Undiluted	10.4
	Diluted 1:10	37.0
	Diluted 1:20	39.1
	Diluted 1:30	39.9
Cell extracts	Undiluted	1.1
	Diluted(1:1)	0.9
	Diluted(1:5)	0.0

ABA Bioassay:

The influence of different concentrations of synthetic ABA, extract, microbial spent medium and water as control on inhibition of cucumber hypocotyls were studied. The excised cucumber hypocotyls were incubated in above mentioned treatments. At the end of the incubation period, inhibition of hypocotyl were measured. The results indicated that both ABA and cell extracts were effective in inducing inhibition of hypocotyl elongation proportional to the levels applied (Table2). The minimum detectable level of abscisic acid was 10^–8 M, and the range of linear response to abscisic acid was shown between 10^–7 and 10^–3 M. The hypocotyls in undiluted methylotroph extracts became soft and decayed whereas in 1:5 dilution of extract more inhibition was observed compared to 10-3 M concentration of synthetic ABA. The hypocotyls treated with 1:10 dilutions of extract showed inhibition level between 10^-3 and 10^-4 compared to the standaeds, which reveals that ABA level in MSF 34 extract is more than 10^-3M, which further quantified through HPLC.

Table 2: Influence of different concentrations of ABA, MSF 34 extract and microbial spent medium on inhibition of cucumber hypocotyls

Treatments		% inhibition over control
ABA (M)	10ˉ³	45.76
	10^-4	40.68
	10^-5	24.04
	10-⁶	17.26
	10^-7	11.87
	10^-8	1.7
	10-9	00.00
Microbial spent medium	Undiluted	decayed
	Diluted 1:10	decayed
	Diluted 1:20	00.00
Cell extracts	Undiluted	Tissue became soft
	Diluted(1:1)	46.38
	Diluted(1:5)	45.94
	Diluted(1:10)	43.02

Quantitative analysis by HPLC

Methylotroph cultures were centrifuged and ABA and IAA were extracted from pellet by using methanol as a conventional extractant. Microbial spent medium and cell extracts were purified by using sterile nylon membrane syringe filters of pore size 0.45µm under aseptic conditions and was subjected to HPLC analysis. The standards used were synthetic IAA and ABA in the concentration of 1mg/ml of ethanol (1000 ppm). Chromatography on the ODS 3V C₁₈ column in MeOH-2% HAc-H₂O (40:40:20) solvents system separation of IAA and ABA could be determined in IAA &ABA standards, microbial spent medium and cell extracts are shown in fig. 2, 4, 3 and 5 respectively. The peaks from microbial spent medium and cell extracts were compared with the peaks of standard IAA and ABA; the supernatant witnessed only IAA which is 3.75 times higher than standard IAA. On the other hand, concentration of ABA in the extracted sample was estimated to occur 4 times more (3922ppm) than the standard used.

Retention time (min)

Fig. 2: HPLC of the standard IAA

Retention time (min)

Fig. 3: HPLC of the standard ABA

Retention time (min)

Fig. 4: HPLC of the microbial spent medium

Retention time (min)

Fig. 5: HPLC of the extracts of cells

DISCUSSION:

In the present work, the presence of phytohormones such as IAA and ABA were detected in the microbial spent medium and cells of the methylotroph MSF34 respectively. Similar results have been reported in microbes of the genus Methylobacterium, maintained in liquid culture, which produce cytokinine and secrete it into the surrounding medium¹⁸. The classical phytohormone, auxin is also produced and secreted by different strains of Methylobacterium^{14,21,1,12,13}. Other species of bacteria reported to produce phytohormones^19,23 are A. xylosoxidans Ax10 and L .fusiformis PM5 & PM24 which produce nearly 5.0 ug /ml and 100 um/ml IAA respectively in defined medium. Recently Verónica Sgroy et al., (2009) observed the production of IAA, cytokinine, GA and ABA in P. putida, A. xylosoxidans, L. fusiformis, B. licheniformis, B. pumilus and B. subtilis and B. halotolerans. Among all these strains P. putida produced the highest concentrations of IAA (2.25 μg ml⁻¹), and ABA (4.27 μg ml⁻¹) in chemically defined medium. Inhibition of plant growth is often used as a basis for the qualitative bioassay of abscisic acid⁷. Dorffling and Tietz (1983), reported cucumber hypocotyl bioassay is a routine bioassay of ABA that has an acceptable sensitivity range, an acceptable inhibition period, range of linear response and reliability within usable range. ABA production within the cell of Bradyrhizobium japonicum (strain USDA110) is also reported⁵. The tissue become soft and dead in un diluted microbial spent medium and cell extracts may be due to higher concentrations of respective hormone presence. . Biosynthesis and secretion of IAA and biosynthesis of ABA by methylobacteria maintained in liquid culture medium was witnessed via bioassays followed by further quantification of the same through HPLC. The hormones abscisic acid (ABA) and indole-3-acetic acid (IAA) in has been isolated and measured by the methods^10,11,15, reversed- and normal-phase HPLC also been used to separate a wide range of gibberellins (GA3) both as free acid and as their methoxycoumaryl esters ¹⁶.

CONCLUSION:

The Methylotroph strain MSF34 has the ability to produce two major phytohormones and this in fact may have two important technological implications 1. Since ABA is expensive this strain can be use for commercial production of ABA. 2. The formulation can be used for the improvement of drought tolerance in crops under water deficit conditions. This leads the way for future studies of the Methylobacterium-plant relationship under drought.

ACKNOWLEDGMENTS:

The authors thank Dr. S. Seshadri, Director (R&D), Shri AMM Murugappa Chettiar Research Centre for providing the culture to carry out the experiments.

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Received on 10.09.2012 Modified on 16.09.2012

Asian J. Research Chem. 5(9): September, 2012; Page 1190-1194