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INTRODUCTION

Plant growth hormone rhizoflora are involved with host plant in mutual interaction. They
promote plant growth by the production phytoharmone, biocontrol of host plant diseases
or improvement of plant nutritional status. The beneficial effect of rhizobia in terms of
biological nitrogen fixation has been the main focus of study in the

past. besides

biological nitrogen fixation, some strains of rhizobium are also involved in plant growth
hormone activity PGPR activity includes production of phytohormone like indole acetic
acid (IAA) and gibberellins, but IAA production was studies in Rhizobia

strains

associated only with a few legume hosts (Basu and Ghose , 2001; Ghose and Basu 2002;
Roy and Basu 2004). It is well known that the Rhizobium strains isolated from single
host sps very in their culture and biochemical characters. Hence the present work was
taken up to study the IAA Synthesizing capacity of different strains of Rhizobium
isolated from root nodules of sesbania (L) Merr. And to find out the culture requirement
for maximum IAA production. Twenty six rhizobium strains were isolated from root
noduls of sesbania sesban (L) Merr. , collected from different regions of Madhya
Pradesh , using yeast extract Mannitol agar medium . The identity of the culture was
established by plant infection test (Vincent, 1970). The strains were tested for their
production of indole compounds in 1% tryptone broth followed by the methods described
by Dubey and Maheshvari (2002). The IAA in culture supernatant was quantified by
growing the cultures in bacteria and fungus extract mineral broth supplemented with Ltryptophan (Bhattacharya and Basu 1992) and estimated by sing colorimetric assay suggested by
Gordon and Weber (1951). Qualitative test showed that all the 26 Rhizobium strains produces
indole compounds 1% tryptone broth. The amount of IAA produced varied from strains to strain
and relatively more amount was observed in Rhizobium strains. The effect of different
concentration of L-tryptophan revealed that the maximum growth and IAA production were
observed in all the five strains (Ilic et al Rhizosphere soil represents a unique Plant biological
niche with a diverse micro flora comprised of bacteria, fungi, protozoa and algae. This
community is supported nutritionally by a high input of organic materials derived from the plant
roots and root that are necessary for microbial growth (Lynch 1990). However, the composition
and quantity of root exudates varies depending on the plant species (Smith1976) and the physical
environment such as humidity and temperature (Martin and Kemp1980). Actinomycetes are
Gram-positive bacteria They are the most widely distributed group of microorganisms in nature,

pg. 1

and are also well known as saprophytic soil inhabitants (Takizawa et al. 1993). Most
Actinomycetes in soil belong to the genus (Good fellow and Simpson 1987, Suzuki et al.2000)
and 60% of the sources of most biologically active compounds such as antifungal and
antibacterial compounds or plant growth promoting substances that have been developed for
agricultural use originated from this genus 2007Streptomycesoccur in the Rhizosphere of plants
and can enhance plant growth by producing plant growth promoter substances. Auxin or
gibberellins (Kaunat 1969, Brown 1972, Merck et al. 1987). The auxins are a group of indole ring
compounds which have the ability to improve plant growth by stimulating cell elongation, root
initiation, seed germination and seedling growth (El-common natural auxin and is a product of Ltryptophan metabolism in microorganisms. Approximately 80% of Rhizosphere bacteria secrete
IAA (Bhavdish et al. 2003). Streptomycin sps inhabiting the rhizospheres of various plants, also
serves as good source of IAA. The rich supply of the potential for the streptomycetes to
synthesize and release IAA (Wheeler et al.1984, Kravchenko et al. 1991, Martens and
Frankenberg 1994). Several Streptomyces species, such as S. olivaceoviridis, S. ramous, S. ruche
and Streptomycin spp. From the tomato Rhizosphere, have the ability to produce IAA and
improve plant growth by increased seed germination, root elongation and root dry weight
(Aldesuquy et al. 1998,Tokala et al. 2002, El-Tarabily 2008).Nowadays, some Rhizosphere
Actinomycetes are studied and developed as a commercial product. For example, Mycostop,
based on the strain K61 of S. griseoviridisandS.lydicusWYEC108 can produce IAA to promote
plant growth (Mahadevan Streptomycin associated with the with the Rhizosphere soil of
medicinal plants has been poorly studied as plant growth promoting agents. These soils may be
attractive sources of Streptomycin

ssp., capable of producing bioactive compounds related to

plant growth promotion (Than gapandian et al. 2007). IAA production of Streptomycin ssp.
Isolated from some Thai medicinal plant Rhizosphere soils and to study the optimization of IAA
production by an active strain, S. viridisCMUH009. The in vivo effect of culture filtrates on soil
sample seed germination and root elongation was evaluated and compared with the commercial
IAA.
Phytohormone;
Phytohormone function to coordinate plant growth and development. The compounds that have
been considered as plant hormones are:
Indole Acetic Acid (Auxin)
Cytokine

pg. 2

Gibberellins
Ethylene
Abscisic acid
Auxin (Indole acetic acid)
Auxin are compounds that positively influence cell enlargement, bud formation and root
initiation. They also promote the production of other hormone and in conjugation with cytokine,
they control the growth of stems , roots, and fruit, and convert stems into flowers , auxins were
the first class of growth regulators discovered .they affect cell elongation by altering cell wall
plasticity, they stimulate cambium a subtype of meristem cells, to divided and in stems cause
secondary xylem to differentiate.

Auxin regulated responses include:
Induction of lateral and adventitious roots, Stimulation of fruits growth, Apical dominance, Leaf
and flowers abscission
Cytokine:
Cytokine or CKs are a group of chemicals that influences cell division and shoot formation. They
were called kinins in the past when the first cytokine were isolated from bacteria cell. They also
help delay senescence off tissue, are responsible for mediating auxin transport throughout the
plant, and affect intermodal length and leaf growth.
ETHYLENE: Ethylene is a gas that forms through the breakdown of methionine, which is in
cells, Ethylene cell and escape out of the plant. Its effectiveness as a plant hormone is dependent
on its rate of production versus its rate of escaping in to the atmosphere.
GIBBERELINS:

pg. 3

Gibberellins, or Gas , include a large range of chemicals that are produced naturally within plants
and by fungi. They were first discovered when Japanese researches including Eiichi Kurosawa,
noticed a chemical produced by a fungus called Gibberellins fujikuroi that produced abnormal
growth in rice plants, Gibberellins are important in seed germination, Affecting enzyme
production that mobilizes food production used for growth of new cells. This is done by
modulating chromosomal transcription in soil sample .Gibberellins also reverse the inhibition of
shoot growth and dormancy induced by ABA.
Rhizobium species:
Lentil has inherent to fix atmospheric nitrogen in association with Rhizobium leguminosarum and
generally gives poor responses to inoculation (Khurana et al 1997) because of the buildup of
rhizobia was probably due to better survival of inoculated Rhizobium sp in Rhizosphere in
presences in soil condition these bacteria although present in soil very in number, effectiveness
in nodulation and nitrogen fixation (Henzell et al 1988). Inoculation of mung bean with
Rhizobium spp. Increased plant height, leaf area, photosynthesis rate and dry matter production
(Thakur et al 1995). Soil inhabiting bacteria developing nitrogen fixing symbiosis with legumes
are classically soil sample.

1. Aim and objectives
pg. 4

e. 2002 worked on indole acetic acid production by a Rhizobium sps from root nodules of a leguminous shrub. 4. \ REVIEW OF LITRETURE Datta et al. The IAA production could be increased pg. Cajanus cajan”. Isolation and purification of bacteria from soil rhizoflora Characterization of isolated bacteria from soil.. 2. Batch fermentation by selected bacterial strain to production of IAA Quantitative and quantitative estimation of produced IAA by batch fermentation. Production of plant growth hormone (IAA) by microorganism isolated from rhizoflora soil using batch fermentation methods with following objectives is as below: 1. 3. 5 .With the present study there is keeping in my mind according to present concept i.

Madhuri M. fruit development and senescence. The production of IAA could be increased up to 70% over yeast extract glucose medium by supplement znso4. Hence the present paper discusses isolation of rhizobia from the selected leguminous plants and its IAA production ability under laboratory conditions. from cell division. flowers and fruits. 2002 reported the growth behaviour and indole acetic acid (IAA) Production by a Rhizobium isolated from root nodule of Alsicarpus vagibalis DC. Many bacteria are known to produce IAA. the symbiot was isolated and identified as a Rhizobium sp. The possible relationship between the rhizobia IAA production and legume – rhizobia symbiosis is discussed. The isolated preferred L. flowering. ASPARAGINE(0.” Investigated that from the root nodules of Alsicarpus vaginalis DC.5MICRROG/ML..(IAA) Not only plants but also microorganisms can synthesize auxins and cytokines. NiCl2 (10 microg/ml) and glutamic acid (0. callus formation.2%) AND sodium dodecyl 7H2O (1.) l- (microg/ ml). parthenocarpy and so on. 6 . Sahasrabudhe 2011 Screening of rhizobia for indole acetic acid production. They can also prevent abscission of leaves. The ability to synthesize phytoharmone is widely distributed among plantassociated bacteria. 80% of the bacteria isolated from plant Rhizosphere produce IAA. Naturally occurring substances with indole nucleus possessing growth-promoting activity are referred to as auxins. The bacteria produced a high amount (107 microg/mi) of indole acetic acid (IAA) in culture from tryptophan supplement yeast extract Mannitol medium. elongation and differentiation to tropic responses. chemically it is Indole acetic acid.5g/l).up to 653. The possible role of rhizobia IAA on the rhizobia legume symbiosis is discussed. Auxins are employed to induce rooting. The action and interaction of some growth regulators like auxins regulate most of the physiological activities and growth in plants. Bhattacharyya et al. pg. The production was maximum when the bacteria reached its stationary phase of growth. The phytoharmone auxins play a central role in plant growth and development as a regulator of numerous biological processes.3% over control by supplementing the carbon free incubation medium with glucose (5g/l).isomer of tryptophan for maximum IAA production.

Hassan Etesami et al. After 48 h of incubation. 2009.6 μg/ml) in isolate-3 at 72 h of incubation. viciae) and a non-inoculated control.5. 1 and 4. 7 .5. K Uptake by Wheat Grown under Greenhouse Condition A greenhouse study was conducted to determine the effects of the superior indole-3-acetic acid (IAA) producing Rhizobial and treatments of Ag (Ag ion) and L-tryptophan (Trp) on uptake of N. isolate 2 and 5 (from CV-2 and 5) showed maximum growth on glucose. 10 and 100 µM) were used. Plants inoculated with the rhizobia together Ag ion and L-tryptophan (Trp) gave the highest root dry weight pg.0 and 2. These isolates were further tested for the production of IAA in a medium with 0.1 g/kg soil). Rhizobium strains were isolated from root nodules of five cultivars of Vegan trilobata namely: cultivar-1 from Prakasham District.31 to 7. Isolate-1 and 3 (from CV-1 and 3) grow better in all carbon sources. All the five isolates produced maximum growth in a medium containing 1 mg/ml conc. The effect of different carbon sources on IAA production was studied by replacing mannitol in YEM broth with equal quantities of different carbon sources.Ravi Kumar and Raghu Ram.11. three levels of Ag ion (0. cultivar-2 from East Godavari District.. with maximum acidic pH of 4. 2012. Plants were grown + for 90 days and plant root dry weight and the uptake of N.31.89 μg/ml followed by isolate-3. The strains were examined for production of acid and indole acetic acid (IAA) by utilizing different carbon sources. 2. Isolate-4 (from CV-4) showed maximum growth on arabinose. of L-Tryptophan. 1. cultivar. isolate-3 produced a maximum of 80. 1.5 mg/ml of L-Tryptophan. almost the five isolates produced acid ranging between 4. Five strains with different ability of IAA production (belonging to genus Rhizobium leguminosarum var. Production of indole acetic acid by Rhizobium isolates from Vegan trilobata (L) Verdc. phaseoli and Rhizobium leguminosarum var. Effect of Superior IAA Producing Rhizobia on N. two levels of L-tryptophan (Trp) (0 and 0.0. Maximum amount of IAA was produced (92. P and K were determined. 5. cultivar-3 from Guntur District. Among the isolates. P. whereas. All the five isolates initiated IAA production immediately after inoculation and maximum was produced at 72 h in YEM broth and showed a decline afterwards.4 from West Godavari District and cultivar-5 from Krishna District on yeast extract Mannitol broth (YEMB) medium with bromothymol blue indicator. Isolate-2 produced IAA at a maximum of 82.96 μg/ml of IAA. P and K by + wheat.

Some of Plant growth promoting characteristics such pg. indole acetic acid (IAA) was produced by Ma-G1 and Ra-I2. P and K) by producing IAA and subsequently the increase of plant root system.Therefore. and determination of their potency for growth factor production Isolation and characterization of Rhizobium spp. Five isolates (Ma-G1. Therefore. The experiment indicated that rhizobia could used as bioenhancer and biofertilizer for wheat production and usage of both Ag and L-tryptophan (Trp) treatments together result in a significant increase on the uptake of N. would be the beneficial bacteria to interplay with the growth promotion of leguminous plants along with biological nitrogen fixation. were able to fix nitrogen in media. P and K in comparison with using Ag ion and Trp alone and also + in comparison with the blank. 8 . P and + K compared to non-inoculated control plants. Jakaria Al-Mujahidy 2013. Lo-F1. and determination of their potency for growth factor production The most important step of this study was to isolate Rhizobium species and determination of their potency for growth factor production. two nonleguminous plants and control soil (without plant). 2013. Sh-J1and Ra-I2) were selected as Rhizobium hainanense and better result was regarded in their assessment for production potency of various growth factors. Isolation and characterization of Rhizobium spp. this Rhizobium spp. the rhizobia could uptake more nutrients (N. The exopolysaccharide production rate of Ma-G1 was enhanced expectedly (1. Unfortunately. Yogendra Singh et al. High salt tolerance was also observed by all strains in media supplemented with IAA containing 510% KH2PO4.. Among these isolates. these five strains were unable to separate soluble phosphorus content from insoluble tri-calcium phosphate (TCP).and the uptake of N. We isolated 260 type bacteria on PCA (Plate Count Agar) media from adjacent soil samples of ten leguminous plants. Then pure cultures of 53 nitrogen fixing bacterial strains were isolated on selective Yeast Extract Mannitol Agar (YEMA) medium. Ch-H2. PGPR function in three different ways synthesizing particular compounds for the plants facilitating the uptake of certain nutrients from the soil and lessening or preventing the plants from diseases.25-fold increase) by treating with IAA. All Rhizobium spp. Rhizobium has been noted as PGPR in almost all review articles.

ability to produce ammonia (NH3) as sole nitrogen source and production of hydrogen cyanide were evaluated in three Rhizobacteria isolated from Rhizosphere of pigeon pea. was examined by using radish as a pg. all the isolates were able to produce Indole acetic acid (IAA) and Production of ammonia was commonly detected in the isolates of Rhizobium (66%). the plant growth hormone indole-3-acetic acid (IAA) in a yeast malt extract medium supplemented with 2 mg/mL L-tryptophan.) Bradyrhizobia and rhizobia are symbiotic bacterial partners forming nitrogen fixing nodules on legumes. isolated from some Thai medicinal plant Rhizosphere soils A collection of Streptomyces spp. Potential of Rhizobium and Bradyrhizobium species as plant growth promoting rhizobacteria on non-legumes: Effect on radishes (Raphanus sativus L. 9 . 2010. Hani Antoun et al. The IAA from this strain was extracted. Among these isolates. These bacteria share characteristics with plant growth promoting rhizobacteria (PGPR). They also exhibit antagonistic effects towards many plant pathogenic fungi. Streptomyces CMU-H009 recovered from soil associated with lemongrass (Cymbopogon citrates) was very effective in producing IAA. Based on the 16S rDNA sequence analysis. Nodule inducing bacteria. are capable of colonizing the roots of non-legumes and produce phytoharmone. The potential of nodule inducing bacteria to function as PGPR. isolate YSY-25 were tolerant to all the heavy metals (200 μg ml-1 ). isolated from the Rhizosphere soils of 14 Thai medicinal plants were found to produce. The culture filtrate from the strain CMU-H009 stimulated a significant increase in the germination and root elongation of maize (Zea Mays) and cow pea (Bruguiera parviflora) seeds. like other PGPR. Indole-3-acetic acid production by Streptomyces sp. this strain was most closely related to Streptomyces viridis NBRC 13373T (99. whereas YSY-26 and YSY-27 were tolerant to all heavy metals (50 to 100 μgml-1 Sutthinan Khamna et al.as Indole acetic acid (IAA) capacity.0..3% similarity). 1998. The IAA production was maximums (300 mg/mL) when the strain CMU-H009 was cultivated in a yeast malt extract broth amended with 2 mg/mL L-tryptophan a pH 7. among test isolates In this study. HCN production was observed in 33% isolates. siderophores and HCN. purified and identified by thin layer chromatography.. and incubated at 30oC with shaking at 125 rpm for 3 d.

causing a 44% reduction in radish dry matter yield. The strains differ in their growth and production of IAA on different carbon and nitrogen sources. 10 . Bhattacharyya. performed in growth cabinets. Three percent of the 266 strains tested were found to be cyanogens. Fifty eight percent of the strains produced indole 3-acetic acid (IAA) and 54% solubilized phosphorus. and an arctic strain (N44) was the most deleterious. The compound was extracted. Sesbania sesban (L. All the 26 Rhizobium strains produced indole acetic acid (IAA). Root nodules of Sesbania cannabina contained a moderate amount of indole acetic acid and tryptophan. japonicum significantly increased (15%) the dry matter yield of radish.) Merr.and Disomers of tryptophan for both growth and IAA pg. The isolated Rhizobium produced 250 mg/ml of IAA in the culture when grown in the yeast extract mannitol (YEM) medium supplemented with different isomers of tryptophan. effects of heavy metals on growth and indole acetic acid production by rhizobium SP. the precursor of IAA.5 mg/ml Ltryptophan. Bioproduction of indole acetic acid by Rhizobium strains isolated from root nodules of green manure crop. 2006. Some of the bacterial species examined were found to have a deleterious effect while others were neutral or displayed a stimulatory effect on radishes. collected from different regions of Andhra Pradesh. purified and structurally confirmed as IAA. Cultural requirements were optimized for maximum growth and IAA production. while a majority (83%) produced siderophores. The microsymbiont isolated from the root nodules was identified as a Rhizobium sp.) Merr. Mutluru Sridevi and Konada Veera Mallaiah 2013. Bradyrizobium japonicum strain Soy 213 was found to have the highest stimulatory effect (60%). A second plant inoculation test. Addition of cell wall affecting agents increased the IAA production over controls. Twenty six Rhizobium strains were isolated from root nodules of Sesbania sesbania (L. but maximum amount was produced by only five strains in yeast extract mannitol (YEM) medium supplemented with L-tryptophan. revealed that only strain Tal 629 of B. The strains were found to elaborate maximum IAA when fed with 2. The level of IAA Oxidase was much higher in the roots than in the associated nodules. The roots contained non detectable amount of IAA and very low level of tryptophan. The bacteria preferred L-isomer over DL.model plant. This indicates that specific bradyrhizobia have the potential to be used as PGPR on non-legumes.

Nowadays. All the 26 Rhizobium strains produced indole acetic acid (IAA).0 mg/ml). collected from different regions of Andhra Pradesh.production. siderophores production. To develop local biofertilizer strains. 11 . All the four heavy metals had inhibitory effect on growth and IAA production. Siderophore production and phosphate solubilization were evaluated in selective culture conditions. further work on the effectiveness of metals on resistant and sensitive rhizobial strains (in respect of hormone production and nitrogen fixation) is needed. Indole-3-acetic acid (IAA). and SEMIA5080) of Bradyrhizobium japonicum. The strains were found to elaborate maximum IAA when fed with 2. and phosphate solubilization in three strains (E109. Addition of cell wall affecting agents increased the IAA production over controls. but maximum amount was produced by only five strains in yeast extract mannitol (YEM) medium supplemented with L-tryptophan. Cultural requirements were optimized for maximum growth and IAA production. most commonly used for inoculation of soybean and nonlegumes in USA. Phytoharmone production by three strains of Bradyrhizobium japonicum and possible physiological and technological implications Phytoharmone production by three strains of Bradyrhizobium japonicum and possible physiological and technological implications he aim of this work was to evaluate phytoharmone biosynthesis. besides nitrogen fixation. Twenty six Rhizobium strains were isolated from root nodules of Sesbania sesbania (L. which had negative results. and South America. For growth and IAA production pH 7. Canada. Sridevi and Konada Veera Mallaiah. Boiero. USDA110.5 mg/ml Tryptophan. The effect of Hg. hormone production was also considered as an important aspect in the legume-rhizobia symbiosis.) Merr. particularly at higher concentration (1. Bioproduction of indole acetic acid by Rhizobium strains isolated from root nodules of green manure crop. gibberellic acid (GA3). Pb. 2007. and abscisic acid pg. The strains differ in their growth and production of IAA on different carbon and nitrogen sources. Cd and Ba metals on IAA production by Rhizobium was also investigated and it was found that Hg2+ had the most inhibitory effect. The isolate reached its stationary phase at 24 h for both these functions. The compound was extracted. 2007.5%) were found to be optimum. purified and structurally confirmed as IAA.1 and mannitol (0.

12 . however. their levels were significantly higher (p The aim of this work was to evaluate phytoharmone biosynthesis. and abscisic acid (ABA) production were analyzed by gas chromatography–mass spectrometry (GC-MS). Ethylene and zeatin biosynthesis were determined by GS–flame ionization detection and high-performance liquid chromatography (HPLC-UV). respectively. IAA. and GA3 were found in all three strains. and phosphate solubilization in three strains (E109. and SEMIA5080) of Bradyrhizobium japonicum. most commonly used for inoculation of soybean and nonlegumes in USA. Ethylene and zeatin biosynthesis were determined by GS–flame ionization detection and high-performance liquid chromatography (HPLC-UV). and South America. respectively. zeatin. however. which had negative results.G. USDA110. gibberellic acid (GA3). zeatin. their levels were significantly higher have an important technological implication for inoculants formulation. and GA3 were found in all three strains.(ABA) production were analyzed by gas chromatography–mass spectrometry (GC-MS). siderophores production.) pg. IAA.1REQUIREMENTS: Equipments:  Refrigerator (L. Siderophore production and phosphate solubilization were evaluated in selective culture conditions. MATERIAL AND METHOD 4. Indole-3-acetic acid (IAA). Canada.

sulphonilic acid. aluminum chloride. ferrous chloride.Dipotassium phosphate .2 Chemicals: NAM (Nutrient Agar). ascorbic acid(HIMEDIA). Agar Agar Type I (H. Sodium Hydroxide Flakes . Dextrose anhydrous . . Ferrous sulphate heptahydrate. Methyl Red Solution (Indicator) – Acetone . Tryptone Type – I.2. Ethanol (Ethyl alcohol) . Weighing Machine  Magnetic stirrer (REMI)  Vortex Mixer  Autoclave  Laminar Air Flow  Incubator  Spectrophotometer  Water Bath  Hot Air Oven  Micropipette  Water bath  Soxhlet 4.Potassium Phosphate. Sodium chloride crystal GR . Magnesium sulphate ) silica gel tannic acid. Magnesium sulphate hepta hydrate (Pure) – . Sucrose . phenol Red (Indicator)-.Gelatin(Purified)Sodium nitrate (Purified) ( Potassium chloride (Purified). sodium molibdate. TCA. sodium salicylate. Ammonium dehydrogenate phosphate . Bromophenol blue. . Extrapure . Yeast Extract Powder .  . Dipotassium Hydrogen Phosphate. Potassium Hydroxide. potassium iodide. Starch Agar . Dye and reagents: pg.Pectin . Purified . 13 . Certified (Coomassie Brilliant blue G 250 . Ammonium Peroxodisulphate Extra pure . Skim Milk powder . Potassium ferry cyanide sodium nitropruside. Beef extract . Peptone . Acetic Acid glacial . Methan Litmus .

Glass wares & Plastic Wares: Micropipette Tips. vials. Heat fixes the smears. At each site 10 sample were taken randomly. Eppendorf tubes. Gram’s iodine solution. pg. Crystal violet. Durham tubes. where stone in the sample were removed and the soils were homogenized through a 2mm sieve. test tubes. Butter paper. The samples were then transported in sealed aluminum foil to the laboratory. The sample were designated as rose papaya chameli mango kada Neem sem etc in the dark bottle till further analysis. watsman filter paper. filter paper. cuvets. pipettes..Bromothymol blue. 2011). Forceps. Eppendorf tubes stand. MICROBIOLOGICAL ANALYSIS OF SOIL Isolation and Identification of Microbes The soil samples serial dilutions were Prepared and a volume of 0. Petri dishes. biuret. Other Miscellaneous: Aluminum foil. Kovac’s reagent. Cotton. Hexadecyl trimethyl ammonium bromide. Morphological Characterization: The microbial identification was done on the observation of colony morphology and their microscopic observation. safranine. Conical flask. Tissue paper.P). Method of Gram Staining:    Make thin microbial smears on glass slide.1 ml was spread on pikovskaya medium for the isolation of P solubilizing microbes. Collection of Soil Sample: Soil samples were collected from Rhizosphere of different plant of different location of nearby region of Bhopal (M. The inoculated Petridishes were incubated 48 h on 37°C. spatula. The isolates were maintained on pikovskayas broth for further analysis (Eva Lazlo et al. Beaker. Let the smear air dry. 14 .

Kovac’s reagent 4. Nutrient agar slant cultures 2. 2. Requirements 1. chemicals and kits etc that are to be stored at low temperature. with other with accuracy and also for the measurement of liquids that are harmful if miss handled 3. Refrigerator-Godrage: The refrigerator is must in biotechnology lab used to keep different solution. the indole produced during the reaction is detected by adding Kovac’s reagent (dimethylaminobenzaldehyde) which produces a cherry-red reagent layer as illustrated. Biochemical characterization Some bacteria oxidize tryptophan. Weighing balance: It is an electronic instrument used to weigh definite and accurate amount of required materials.  Wash each slide with distilled water for a few seconds. Apply safranin to smear for 30 seconds.  Hold the smears using slide rack or cloths pin. Using that we can the materials unto five places of decimal. Tubes containing 1% tryptone broth 3. until no more color flow from the smear. Bunsen burner 1. The indole test is performed by inoculating a bacterium into tryptone broth. 4. Micropipettes and tips: These are used for the measurement of littlie amount of liquid such as in micro liters which cannot be measured. using wash bottle. an essential amino acid. Cover each smear with crystal violet for 30 seconds. Dropper bottle 5. Magnetic stirrer and hot plate: pg. 15 . Wash with distilled water and blot dry with absorbent paper. Wash the slide with distilled water and drain. add ethyl     alcohol drop by drop. Let the stained slides air dry.  Wash off the iodine solution with 95% ethyl alcohol.  Cover each smear with iodine solution for 30 seconds. pyruvic acid and ammonia. by the enzyme tryptophan’s resulting in the formation of indole. 1ml Pipette 6.

Weighed amount of ingredients was put in 500 ml of distilled water. More distilled water was added to make up 1 lt. aluminum foil spatula butter paper cotton forceps and tissue paper vials culture tubes. as the case may be. pg. by adding either acetic acid or alkali.0) Peptone 05. Ph meter/strips PH strips are used to adjust the ph of the solution as required. It is also used as a heating plate. Petri plate water solution etc.Magnetic stirrer with hot plate was used to stir the components of solution or to dissolve any liquid. 15 lbs pressure for 15 minutes. Autoclave Autoclave was used to for sterilization of different material in the such as tips. Constituents were dissolved by heating. Autoclaved media was allowed to cool.0 gm Beef extract 03. 7. Glassware & plastic wares Micropipette tips beaker flask.7 using a Ph meter. 5. The media was autoclaved at 121º C. It needs to be standardized by using two buffers one ph-4 and other with ph-7 6. Media was store at room temperature for future use. pH of the medium was adjusted to 7. 8. 3. Media used for bacteria: Nutrient Agar Media (NAM) (pH 7.0 gm Agar 15. 16 . 151 lbs pressure. 5. 4.0 gm Sodium chloride 05. 2. 6. 7.0 gm Distilled water 1000 ml Procedure: 1. 121˚temperature from 15 minutes. Cotton plug was applied and rap it with brown paper.

85 gm NaCl was weighed and dissolved in 100 ml distilled water. Vortexes it for 5 min. Vortex machine Procedure: 1. Plating (Inoculation): pg.B. 3. 5. 1 ml of suspension from tube 1 was pipette into tube 2 with a sterile pipette under aseptic condition to make 1:100 (10-2) dilution and vortexes it for 5 min. 17 . Requirements: 1. Allowed it cool at room temperature. On direct culturing from sample smear is formed. 0. 1ml of sample suspension was pipetted in sterilized test tube 1 having saline water to make 1:10dilution (10-1). Test tubes 2. For studying them they are cultured on plates in laboratory. Sodium chloride 3. 9 ml of above saline water was pipette in 9 test tubes. 10-4. C. Sterile pipette 4. Serial Dilution of Sample Innumerable microbes are present everywhere in the environment. 8. As a result isolated colonies are formed helps in studying their morphology and in further works. Further dilution of 10-3. 5 gm of sample was dissolved in 10 ml of sterile distilled water by shaking it on magnetic stirrer. Thus. serial dilution of the sample is prepared to reduce the microbes. and 10-5 up to 10-9 was made by pipetting 1 ml of suspension into additional tube as done earlier. 2. 6. Distilled water 5. 7. All the 9 test tubes were autoclaved at 121ºC .15 lbs pressure for 15 min 4.

the inoculums was spreader on plate by rotating it. 3. 4. The plate was incubated at 37ºC in an inverted position for 24 to 48 hours. averaged and multiplied by the dilution factor to find the number of cells / spores per gram (or milliliter) of the sample: No. Pipette 4. The spreader was dipped in the spirit and flamed. of cells/ml or gm = Colonies * Dilution factor Dry wt. elevation. Requirements: 1. Colony Count: Microorganisms are identified initially on the basis of their colony morphology. Serial dilution of sample 2. On cooling of the spreader. In this technique a dilute sample having mixtures of microorganisms are present is plated on basic media so that individual colonies can be isolated. The media was poured on the sterilized petriplate and allow it to solidify. forms.1ml of each serial dilution of sample was added on the nutrient agar plate. Different characteristics of colony which are considered are its color. etc. Spreader Procedure: 1. of sample Dilution factor = Reciprocal of the dilution (e.g. Total colony count of each plate is important for determining the microbial diversity of the sample. 2. The number of colonies appearing on dilution plates are counted. 0.Plating is an initial step of isolating microorganisms from the given sample. 5. 10 -7 = 107) pg. margin. Nutrient agar plate 3. In food industries total count or CFU count of microbes is used as standard for the quality assurance of the foods. D. 18 .

19 . and (iii) spread – plate. Master cultures on nutrient agar 2. Each of these new cultures represents the growth of a single species and is called a pure or stock culture. Sub culturing is the term used to describe the procedure of transferring of microorganisms from their parent growth source to a fresh one or from one medium to another. After plating the next step are to subculture some of the cells from one of the colonies to separate agar or nutrient agar slants with a sterilized needle or loop for further examination and use. 2. Pure Culture Technique Pure cultures of microorganisms that form discrete colonies on solid media may be most simply obtained by one of the modifications of the plating method. (ii) pour plate. 2001) 1. the term picking off is used.Requirements: 1. Each viable organism gives rise. Inoculating loop 4. 3. through growth. When the transfer is done from a solid medium (agar) to a liquid medium (broth). Incubated nutrient agar plate 2. Nutrient agar slants or nutrient agar plates 3. Permanent marker Procedure: (Aneja. to a colony from which transfer can be readily made. Requirements: 1. Marker pencil (pen) Procedure: pg. The three most commonly used methods for obtaining cultures of microorganisms are (i) streak – plate. II. 4. The total number of colonies present per plate was also counted. CFU count of each dilution was calculated. This method involves the separation and immobilization of individual organisms on or in a nutrient agar medium. The number of colonies of same morphology present in each plate was counted. After 24 hours incubated nutrient agar plate were observed.

the dye crystal violet is selectively bacteriostatic for gram positive bacteria and favors enteric pathogens. The selective action is brought by the addition of certain chemicals in the medium e. 20 . 5. thus facilitating bacterial isolation. Petri plates 3. The inoculating loop was again heated to destroy existing organisms. Inoculating loop pg. 4. Selective media a. which permit the growth of some of specific group of organisms while preventing or retarding the growth of other. Identification: A. The loop was allowed to cool for a few seconds or cooled it by dipping in a fresh agar plate. Incubate the cultures Petri plate at 25º C for 48 – 72 hours. A differential medium is that which will cause certain colonies to develop differential from other certain colonies to be develop differentially from other organisms present by producing a characteristic change in the bacterial growth. 2. Mannitol agar c. The inoculating loop was sterilized by holding it in the hottest portion of the Bunsen burner flame until the entire loop becomes red hot. Later the culture was sub cultured in the nutrient agar slant. Requirements: 1.g. Mac Conkey agar 2. 6. The tip of the loop was touched to the surface of a selected discrete colony on the agar spread plate. 3. the nutrient agar slants and agar plates was labeled.1. With a marker pen. The agar plate was inoculated by drawing it lightly over the hardened surface in a zig – zag line. 8. 7. Growth on Selective and Differential Media: The selective media are those. EMB b. III.

3. Spirit lamp Procedure: 1. a differential stain was developed by Dr. 4. 6. Weighed amount of ingredients was put in 500 ml of distilled water. Spirit lamp Procedure: 1. 5. Dissolved the constituents by heating and agitating. Dissolved the constituents by heating and agitating. Inoculating loop 4. whereas those that lose the crystal violet and counter stained by safranine (appears red) are referred to as gram negative. B. Hans Christian Gram. 2. as the case may be. by adding either acetic acid or alkali. The plates was streaked with pure culture and incubated at 37º C for 24 hours. It is a very useful stain for identifying and classifying bacteria into two major groups: the gram positive and gram negative. Petri plates 3. 15 lbs pressure for 15 minutes. not decolorized when stained with Gram’s Method) are called gram positive. Weighed amount of ingredients was put in 500 ml of distilled water. The bacteria which retains the primary stain (appears dark blue or violet) (i. 8. 21 . iodine solution (mordant). Cotton plug was applied and rapped it with brown paper. Autoclaved at 121ºC. the fixed bacterial smear is subjected to four different reagents: crystal violet (primary stain). 9. pg. 7. The media was poured in the Petri plate and allowed it to be solidified.4. The differences 2. alcohol (decolorizing agent) and safranine (counter stain). More distilled water was added to make 1 lt. Allow to autoclave to cool. pH of the medium was adjusted using a pH meter.e. in 1884. Staining: The gram stain. 2. In this process.

22 . 9. Peptone yeast extract broth (containing 0. pH of the medium was adjusted using a pH meter. as the case may be.3. Incubate the broth at the 35˚C For the process of fermentation.2%.0. 2. particularly pharmaceuticals from natural sources such as animal or plant tissue or pg. FERMENTATION: Fermentation is a biological process that converts sugar to acids. 7. 4. The media was poured in the Petri plate and allowed it to be solidified.0%6) concentration.0. 15 lbs pressure for 15 minutes. Now inoculate the medium with the help of inoculating loop using Rhizobium microbes from the agar slant 4. gases. Autoclaved at 121ºC. Allow to autoclave to cool. Divided it in different bottles and sterilized it by autoclaving it for 15 min. or alcohol fermentation takes places both in aerobic and the anaerobic condition. 5. 0. The energy is generated by glycol sis fermentation pathway.4%. More distilled water was added to make 1 lt. Cotton plug was applied and rapped it with brown paper.6% tryptophan) 2. by adding either acetic acid or alkali. Inoculating loop 3.4%.2%. 8. 6. 3. Downstream processing: Downstream processing refers to the recovery and purification of biosynthetic products. It refers to the bulk growth of microorganism on a growth medium. The plates was streaked with pure culture and incubated at 37º C for 24 hours. Bunsen burner Procedure: 1. Prepare peptone yeast extract broth at different concentration of tryptophan containing (0. Requirement: 1.

Then go for the qualitative and quantitative estimation Qualitative estimation of IAA: Thin layer Chromatography (TLC): Thin layer chromatography consists of a stationary phase immobilized on a glass or plastic plate. hormones and antibiotics and enzyme vaccines used in Diagnosis industrial enzyme. After evaporation collect the produce IAA and it derivatives 7. 4. and an organic solvent. including the recycling of salvageable components and the proper treatment and disposal of waste. 3. 23 . 4. generally in marketable quantities while analytical biosepration refers to purification for the sole purpose of measuring a component or components of a mixture and may deal with sample sizes as small sizes as small as a single cell Requirement: 1. Then shake vigorously and allow at the separation of phase. pg.fermentation broth. The constituents of a sample can be identified by simultaneously running standard with the unknown. 3. up to Ph-3 by using 6N HCL. 6. 2. It is an essential step in the manufacture of pharmaceuticals such as antibiotics. The bottom edge of the plate is placed in a solvent reservoir and solvent moves up the plate by capillary action. and natural fragrance and flavor compound Downstream processing implies manufacture of a purified product fit for a specific use. Then pour the acidified filtrate in separating funnel. After the process of fermentation filter the broth contacting microbe with the help of filter paper 1. Collect the lover phase in a petriplate and keep the petriplate at the room temperature to evaporate the diethyl ether. 5. Discard the cell debris present in the filter paper and acidify the filtrate 2. either liquid or dissolved in a volatile solvent is deposited as a spot on the stationary phase. the sample. Add equal volume of diethyl ether into the funnel containing the broth. Separating funnel Diethyl ether Measuring cylinder Petri plates Procedure.

or a beaker with a watch glass on the top pour solvent into the chamber to a depth of just less than 0.5 cm. Bioassay: pg. the different components in the mixture move up the plate at different rates due to differences in their portioning behavior between the mobile liquid phase and the stationary phase. you can line part of the inside of the beaker with filter paper. And kept for the 5 min at. Solvent preparation for TLC: Isopropanol: ammonia: water (16:3:1) STEP 1: Prepare the developing container for TLC The developing container for TLC can be a specially designed chamber. jar with a lid. swirl it gently. and allow it to stand while you prepare your TLC Plate. the wider it needs to be. Prepare the TLC plate. It has done by using the Salkowski’s reagent.When the solvent front reaches the other edge of the stationary phase the plate is removed from the solvent reservoir. Handle the plates carefully so that do not disturb the coating of adsorbent or get them dirty Quantitative estimation of IAAQuantitative estimation of IAA has done for find out the quantity of produced during the process of fermentation. The separated spots are visualized with ultraviolet light or by placing the plate in iodine vapor. Room temperature after that absorbent has been taken by using UV spectrometer at 530nm compare with the standard IAA. Cover the beaker with a watch glass. to aid in the saturation of the TLC chamber with solvent vapors. 24 . After taking the absorption graph has been plotted to determine the quantity of IAA produced. TLC plates used in the TLC as 5cm × 20cm EACH large sheet in cut horizontally in to plates which are 5cm tall by various: the more sample plan to run on a plate. The reagent is mixed with the isolated IAA at the different concentration.

and some colonies may fuse. RESULT AND DISCUSSION Microbial Analysis: Total plate count: The isolation process is a procedure of isolation the mixture of colonies to a single colony. 25 . This process was done by using streaking method to obtain pure cultures. the germinated seeds were counted and the per cent germination was computed by using the formula No of seeds germinated Germination percentage = ------------------------------x100 No of seeds sown Seedling length (cm): On eighth day of germination test. these samples take about three until four days to growth on the plate. ten normal seedlings were taken out carefully at random from each treatment and measured from the tip of primary root to the tip of apical shoot. So. Table. This solution samples were diluted up to 10 -3.For germination test. the diluted samples were transferred into nutrient agar plate and the bacteria were grown on it. From the observation. pg. On crowded plates some cells may not form colonies. the samples need to be diluted. to obtain the appropriate colony number. The soil’s sample was added with 10 ml pure water to obtain solution sample before transferred into nutrient agar plate. By using spread plate method. On 8th day. leading to erroneous measurements. A total of 9 microbial isolates were obtained from the analyses of soil samples taken from different area of Madhya Pradesh through soil dilution agar plating. It is important that the numbers of colonies developing on the plates are not being too large. gram seeds were dipped in broth of bacterial isolates for ten minutes and kept on germination paper.2 Colony morphology and count of sample. The average length of ten seedlings was calculated and expressed as mean seedling length in centimeters.

8 B2 NEGATIVE(-)Rod shaped NEGATIVE(-)Rod shaped NEGATIVE(-)Rod shaped NEGATIVE(-) Rod shaped NEGATIVE(-) Rod shaped Gram positive(+) rod shaped Gram positive(+) rod shaped B3 B4 B5 F1 F2 F3 Fig.3 0. colony of cells/ml) Color Margin Form Elevation 10-3 Brown Entire Circular Flat 37 37 X103 10-3 Green Smooth Irregular Raised 28 28 X103 10-3 White Entire Circular Raised 72 72 X103 10-3 White Locate Irregular Raised 53 53 X103 10-3 White Entire Circular Raised 46 46 X104 10-3 White Locate Irregular Raised 21 21 X103 10-3 White Entire Circular Raised 39 39 X105 STAINING Gram staining of all the isolated bacteria from sample of soil isolates has been done.5 0. Thus.1 Results of gram staining of bacterial culture isolated from soil sample S.6 0.1 Table. 1 Gram staining Biochemical characterization of bacteria and fungi: pg.7 0.1 Bacterial ID B1 Status NEGATIVE(-)Rod shaped 0. the results of gram staining of the pure culture isolated from sample of soil bacteria is listed below in the table no.Dilution Colony morphology Total CFU (No. 26 .4 0.2 0. 0. no.

The result confirms previous findings that the white pigmented colonies can only grow pg. 27 . Unknown microorganism has white pigment colonies on the plate. Pseudomonas and Bacillus. It grows better on ENA plate because the medium is rich thus amplifying the production of the colonies.TEST B-1 B-2 B-3 B-4 B-5 B-6 B-7 F-1 F-2 F-3 Gelatin test Starch + + _ + _ + + _ + + _ + _ _ _ _ _ + _ + hydrolysis Indole + + + + + + + + + + production Citrate test Urease test Catalase + + + + + + + _ _ + _ + _ _ + _ _ + _ _ + _ _ _ _ + _ + + + _ _ _ _ _ _ _ _ _ _ _ _ _ _ + + test Amylase test Cellulose _ _ _ _ _ _ test Fermentati _ _ _ + + on of + _ _ carbohydra te MR and VP + _ + _ + + _ _ _ + + _ _ + _ + + + test Cashien test + _ The micro flora of soil founded in Soil bacteria is Rhizobium spp. Which founded in the entire samples. The discrete colonies are streaked into another NA slants and incubated at 25° C and 37 ° C. Observation on the streak plate isolation after incubation period shows that unknown microorganism 9 only grow in medium supplemented with nutrient agar and enriched nutrient agar at 25° C but not at 37 °C..

(Bergey 2003) Amylase production test was shown positive by R. R.loti and R. Whereas R. the acid production is accompanied by the evolution of Bacilus. as starch hydrolysis around the line of growth of each organisms.legamanosarum shows positive result for Oxidase test. In glucose. A phenol red indicator change colors into yellow colors which mean that carbohydrates have been fermented with the production of acidic wastes.meloti.loti.at 25° C.legamanosarum. Based on this result.meloti the gelatin is liquefied.legamanosarum. (Bergey 1994 Oxidase test is positive for unknown microorganism because there is change in color after 10-15 minutes of this test.loti and R. from R. (Bergey 2003) The pectin’s test was shown negative by R. This test stated that R. the possible unknown has been narrowed down into 6 from the possible species given.meloti as R. The pH indicator changes color into yellow which proofs that fermentation has occurred.legamanosarum shows positive result. The carbohydrate fermentation test proves that unknown 6 is a sucrose fermented. Unknown is a slow growing bacteria thus it takes more than 24 hours incubation period for the colonies to appear in the slants.legamanosarum as Bacillus puluifaciens shows negative result as color changes from red to yellow. R. puluifaciens shows positive result for Glucose fermentation.meloti.loti and R. R. Gram staining showed that the bacteria are Gram negative bacteria because the cells are pink in color under the compound microscope after staining. In glucose. R.meloti.loti and R. However. (Bergey 2003) The Methyl–Red and Voges Proskauer tests differentiates Rhizobium spp. In sucrose. the test on lactose contradicts results found earlier where the pH indicator doesn’t change into yellow but remain red in pg. It has a rod shape with irregular clusters arrangement of colonies. 28 . (Bergey 2003) In carbohydrate fermentation test is positive for glucose and lactose fermentation. (Bergey 1994) The gelatin test further differentiates R. Whereas Bacillus subtilis shows negative result.loti from R.legamanosarum shows negative liquefaction whereas in R.loti.meloti and R. R. there are no bubbles trapped in the tube. R.meloti. the formation of bubbles is seen in the Durnham tube.

: Pectin test pg. : Methyl reductance test Fig. The possible assumption is that lactose fermentation has not occurred yet because the bacteria are slow fermented.color. Fig. 29 . : Vogues pros cure test Fig. : Gelatin production test Fig. : Amylase production test Fig.

: production of hydrogen sulfide.Fig :-Casein hydrolysis a) positive b) negative Fig. b) negative Fig:.Cellulose hydrolysis a) positive b) negative pg. 30 . a) positive.

Glucose fermentation E. S.Fig:.Urease Test Fig. growth on selective and differential media and biochemical analysis the selected isolated bacterial from samples of soil are identified using the software PIBWIN-2007. The results of analysis are referring in table no. No. Bacterial ID IDENTIFIED BACTERIA B-1 Negative rod shaped Pseudomonas sps B-2 Negative rod shaped Pseudomonas sps B-3 Negative shaped Rhizobium sps B-4 Negative rod shaped Rhizobium sps B-5 Negative rod shaped Pseudomonas sps F-1 Negative rod shaped Pseudomonas florescent F-2 Positive mix culture rod shaped Bacillus species pg.04: --Identification of isolated culture from sample.4 Table ..Citrate test Fig:. Microbial Identification Using Software On the basis of results of the gram's staining. 31 .

F-3 Positive rod shaped Bacillus cerus IAA Productuion: Out of 9 microorganisms only 3 bacteria shows positive result for IAA production on selective medium.309 and 0. RF value of produced content 0. 0. S-3 respectively.278. Sample Rf Value 1 S-1 0.No.309 3 S-3 0. S-2. 32 . S.411 Table RF value of produced IAA pg.278 2 S-2 0. Qualitative analysis by TLC: Qualitative analysis had done by TLC (thin layer chromatography) using Petroleum: Ethyl Acetate (8:2) solvent system.411 in produced IAA by selected bacterial isolates S-1.

Fig.64 4 80 µg/ml 0.3872 2 40 µg/ml 0.16: TLC of produced IAA observed under U.768 5 100 µg/ml 0. S.m.V. 33 .Quantitative estimation of auxin produced by selected bacterial species Quantitative analysis was carried out by using UV spectrophotometer at 535 n.NO. So the good quantity of product recovery analyzed by this method.498 3 60 µg/ml 0.No.89 Fig: 2. Standard Standard curve of IAA 1 20 µg/ml 0. in this I observed the good quantity of produced auxin after down stream processing. Sample Root Shoot Root length length hair pg. Light Quantitative analysis of IAA: S.

3 2. B-1. 34 . B-5 9. F-3 11 5. F-2 12 5. B-3 7 2 4 4.3 8.3 4 8. 1.in cm.6 10. B-4 16.8 8. Control .6 15.6 10.3 8.6 5. Standar 19. F-1 5. 9 6.6 3.5 8.6 9.6 8 6.3 5. B-7 5 3. Bioassay of produced IAA with standard: root hair 20 18 16 14 12 10 8 6 4 2 0 root hair : pg. B-6 15.66 15 9 19 d 12. B-2 6 4.6 3 8.33 4.3 4.3 6 12 7.6 11.

35 .shoot length 10 9 8 7 6 5 shoot length 4 3 2 1 0 pg.

Figure 12 Shoots length & stems thickness of control and test plant. pg. 36 .

S2 and S3 are analyzed using various physicochemical parameters as well as microbial analysis has also been done. further research to ascertain the efficacy of the best isolates in situ and mycorrhizal or plant association is certainly needed. IAA has many different effects. the finding of the present study highlighted that IAA. such as inducing cell elongation and cell division with all subsequent results for plant growth and development.producing bacteria from Rhizosphere soil can be easily isolated and may be exploited for local agriculture use. However. I found the good quantity and quality of auxin produced by rhizoflora of soil that was indole 3 acetic acid. as all auxins do. pg. S1. I found the good quantity and quality of auxin produced by rhizoflora of soil that was indole 3 acetic acid. Three samples of rhizoflora of soil.Figure 13 Comparison of roots of control and test plants Conclusion: Samples of rhizoflora of soil were analyzed using various physico-chemical parameters as well as microbial analysis has also been done. 37 .Trp in culture medium. This study indicates that Rhizobium spp. such as inducing cell elongation and cell divisions with all subsequent results for plant growth and development. In conclusions. IAA has many different effects. Particular possesses the ability to produce high IAA with. This and the other isolates have potential for use as plant biofertilizer or bioenhancer for the plant growth improvement. as all auxins do.

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1991 A prototype model for indole3.acetic acid (IAA) Production by azopillum brasience. Snets. 40 . pg.