INTRODUCTION

Indoel-3-acetic acid (IAA) is the principle form of “Auxin”. Which

is a important member of phytohormone.
The plant hormones are designated as “Phytohormones” by Thimann
(1948) in order to distinguish them from hormones. “The phytohormones are the
organic compounds produced naturally in higher plant, controlling, growth other
physiological functions at a site remote from it’s place of production and active in
minute amounts”. Plant hormones are not nutrients but chemicals that in very low
concentration controls the growth activities in plants.
“Auxin” (auxein = to grow or increase) (Kogl and Haagen-Smit
1931) an organic compound or substance which promotes growth i.e. (irreversible
increase in growth) it influence the development and differentiation of cells &
tissues along the longitudinal axis when applied in low concentration to shoots of
the plants.
The plant hormones are recognized as in five major classes

1) Auxins
2) Gibberllins ( Gibbrllelic acid)
3) Cytokinins
4) Abscisic acid
5) Ethylene
Auxins derive their name from greek word “auxono” means to grow.
They were the first of the major plant hormone to be discovered and are major co-
ordinating signals in plant growth and development. In every case an exact
biochemical knowledge of the mechanism of hormone action is unknown. They
study of plant hormones has been carried out mainly at the physiological role.

1
 The most important member of the auxin family is indole 3-acetic
acid (IAA). It generates majority of auxin effects in plant & the most ‘potent
native auxin. The principal naturally occurring auxins in plants have been
identified isolated, purified and their chemical structure determined are all indole
derivatives as mentioned below.
1. Indole-3-acetic acid (IAA)
2. Indole-3-acetonitrile (IAN)
3. Indole-3-acetaldehyde (IAC)
4. Ethylindoleacetate.
5. Indole-3-pyruvic acid (IPYA)
6. Indole-3-ethanol (IETOH)
Besides these natural auxins mentioned above several small organic
molecules have been synthesized which show biologic properties characteristics of
Indole-3-acetic acid (IAA) though not in all respects. They are usually derivatives
of benzoic acid, Indole-3-acetic acid at naphthalene acetic acid. Some of the potent
synthetic auxins are, TIBA, R-4-D, 2-3-S.T., NAA and NOA, phenylacetic acid
(PAA) is however a weak auxin.
The structure of Indole-3-acetic acid is

Indole-3-acetic acid (IAA)

2
 The molecular formula of Indole-3-acetic acid generates majority of
auxin effects in the intact plants thus play important role in water. It has melting
point of 168-1700C.
The Indole-3-acetic acid generates majority of auxin effects in the
intact plants & thus play important role in plant growth and development and it
has large no. of commercial agricultural important as a weedkiller (The amount of
auxin applied in weedkillers is far greater than the amount produced within the
plant).
The effects generated by Indole-3-acetic acid are as
1) Stimulation of cell elongation
2) Stimulation of cell division
3) Induction of formation and organization phloem & xylem.
4) Cambial activity
5) Callus formation and galls
6) Rooting of stem cutting (formation of adventitious roots)
7) Apical dominance
8) Delay (or inhibition) of abscission of leaves.
9) Flowering
10) Fruiting
11) Increase in Respiration
12) Increased Resistance for frost damage

Diverse soil micro-organisms including bacteria, fungi and algae are

capable of producing physiologically active quantities of auxins, micro-organisms
inhabiting rhizospheres of various plants are likely to synthesize and release auxin
as a secondary metabolite. Which may exert pronounced effects on plant growth
and development / establishment various micro-organism inhabiting plant roots

3
and influence the plant growth promoting rhizobacteria (Kloepper et al. 1986;
Fankenbeger & Arshand 1998, Arshad & Frankbeger 1998).
Numerous soil M.O. like Azatobacter pseudomonas, Rhizobium, &
fungi, like Fusarium, Aspergillus, Rhizopus and some actinomycetes produce
Indole acetic acid.
Rhizobial bacteria are the best plant growth promoting among, the
Rhizobacteria. One of most important way that those bacteria affect growth &
development is by producing Indole-3-acetic acid (IAA) that this hormones led to
plant root system development and subsequently nutritional uptake increase by
plant. Many of rhizobial species enable to produce IAA, the bacteria use
Tryptophan (L-Trp) as a precursor, this substance can be used converted to IAA
by soil beneficial bacterial activities.
Thimann in 1936 proposed that Auxins, such as indole-3-acetic acid
(IAA) play a role in root nodule organogenesis. Nitrogen fixing Rhizobia enter
symbiosis with (mainly) leguminous plants eliciting root nodule development
triggered by the Nod factor (Staygard 2000). Several lines of evidence suggest a
role for phytohormones in root nodule development (Fang and Hirsch 1998 &
1992). Early interest in the production of indole-3-acetic acid by Rhizobium was
stimulated by speculation that such biosynthesis might be a necessary part of root
nodulation process (Thimann 1936). There is now strong evidence from Gas
Chromatography (GC) mass spectroscopy (MS) analysis of culture filterates for
IAA Biosynthesis by Rhizobium. It grow in yeast extract mannitol broth &
produce IAA. Although Rhizobium synthesizes IAA in media free of tryptophan,
production is stimulated greatly by supplementation with the amino acid. E.g.
Rhizobium legumenosarum cultured in 100 mg l-trp produced almost 1 mg of IAA
(Wang et.al. 1982).

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 AIMS & OBJECTIVES

Aim : Production of Indole 3-acetic acid from root nodules of Cicer

aerietinum & its effects of germination of seeds.

Objectives :

1. Isolation of Rhizobium species.

2. Identification of Rhizobium Species.
3. To check ability to produce IAA.
4. Optimization of L-TRP concentration.
5. Production of IAA.
6. To check effect of Incubation period on production of IAA.
7. Extraction of IAA.
8. Effects on germination of seeds.

5
 REVIEW OF LITERATURE

The presence growth regulating hormone in plants was first

suggested by Julius Von Sachs in 1980. He proposed that these were certain
‘Organ forming substances’ in plants which were produced in leaves &
translocated downward in plant body. Also in 1980 Charls Darwin studied the
effect of light on plant movements. He demonstrated the presence of transported
signal originating from plant apex, able to promote differential cell elongation in
seedlings lower parts resulting in it’s bending towards the light source (Darwin &
Darwin 1880). This signal was subsequently isolated and named auxin by Wont
(1935). Darwin experiment expanded upon Theophil Ciesielski’s research
examining roots bending forward gravity (Ciesielski 1872). The term auxin was
coined, by scientists in human urine named auxins A & B (Kogl and Haagen Smit,
1931). A structurally distinct compound with auxin activity isolated from fungi
was called heteroauxin; auxin A & B were gradually abandoned for the
reproducibly bioactive heteroauxin which as later determined to be Indole-3-acetic
acid (IAA).
Indole-3-acetic acid (IAA) is the principle form of auxin, which
regulates several fundamental celluar processes, including cell division (Kendre &
Zeevaarl; 1997) elongation and differentiation. It also leads to decrease in root
length and increase in root hair formation, thus enhancing the capability of the
plant to absorb soil nutrients. Besides there are many developmental processes in
which auxin plays a role including embryo & fruit development organogenesis
Vascular tissue differentiation (Aloni, 1995) root patterning elongation & tropistic
growth, apical hook formation (Yang et. al.; 1993 & Kendre & Zeevaast; 1997) &
apical dominance. (Tomas;1995).
Many naturally occurring compounds that exert auxin like effects
have been revealed by this bioassay. A chlorinated form of IAA with high auxin

6
activity, 4-C1-IAA, is found in several plants (Slovin et al.1999). In addition to
the indolic auxins, phenylacetic acid (PAA) has been identified in plant and is an
active auxin (Wighman 1977; Ludwig-Musler and Cohen, 2002).
Certain IAA precursors, such as indol-3-acetonitrile & Indol-3-
pyruvic acid are also active in bioassay, presumably because of conversion in the
tissue to IAA (Thimann-1977). Similarly indol-3-butyric acid (IBA), identical to
IAA except for two additional methylene groups in the side chain, is effective in
bioassay, like IAA exogenous IBA inhibits Arabidopsis root elongation (Zolman
et. al., 2000) and adventitious roots formation (King & Stimort, 1998) IBA,
originally classified as synthetic auxin, is infact on endogenous plant compound
(Epstein & Ludwing Muller, 1993; ludwing-muller, 2000, Bartel et al., 2001). IBA
is more effective than IAA. At lateral root induction, perhaps because, unlike IAA,
IBA efficiently induces-lateral roots at concentrations that only minimally, inhibit
root elongation (Zolman et al., 2000) IBA is employed commercially for this
purpose (Hartmann et al.,1990). Biochemical analyses in a variety of plants &
genetic studies indicates that IBA acts primarily through conversion to IAA in a
process resembling peroxisomal fatty acid B-oxidation (Bartel et al., 2001) though
roles for IBA independent of conversion to IAA have been proposed (Ludwing
muller, 2000; Pouport and Waddell 2000).
The concentration of auxin is critical to the physiological response
with an excess or on deficiency having a characteristics effect. In addition to plant
factors that influence the level of auxin such as de novo, synthesis, auxin secreted
by microbes can contribute to plants endogenous pool. Production of auxin is
widespread among plant associated bacteria, several different biosynthetic
pathways are used by these bacteria, with a single bacterial strain sometimes
containing more than one pathway. The level of expression of IAA depends upon
biosynthetic pathway; the location of genes involved, either on chromosonal or
plasmid. DNA, & their regulatory sequences, and the presence of enzymes that

7
can convert active, free IAA into an inactive, conjugated form. The role of
bacterial IAA in stimulation of plant growth & phytopathogenesis is considered.
Some of the organism are psychopathologic while others promote plant growth;
the auxin they produce is often implicate in their effect on host plant e.g. Auxin
believed to play a role in formation of crown gall tumor by Agrobacterium
tumicfaitians & in stimulation of root development by Azospirillum species.

EXTRACTION OF AUXINS:
The two forms of auxins (free and bound) appear to be in a dynamic
state as there are many examples where the bound auxin is released in free site
during extraction. With the result, the strictly separate measurement of free and
bound auxins is often difficult. There are, however, two methods commonly
employed for auxin extraction.

A. Diffusion method:
It was devised by Went (1928) at Utrecht. In this method the
growing tip (or other organ to be tested), under condition of low transpiration, is
severed (or cut) and is then placed on an agar block (usually of 1.5 concentration)
for about an hour or so. During this period, the auxin diffuses from the cut tip into
the agar block.

(a) Excessive transpiration may prevent the accumulation of the auxin in

the agar block.
(b) Severing the tip results in lowering the amount of auxin from the cut
surface.
(c) The method cannot be widely adopted on account of the presence of
growth inhibitors in many green plants.

8
B. Solvent extraction method:
Here, the tissues are grinded in some organic solvents like
chloroform, ether, ethyl alcohol or even water and the liquid is then filtered. The
auxin is separated from the filtrate by chromatographic technique.
This method is widely employed for the extraction of auxin, esp., the
bound auxins. But this one also suffers from certain drawbacks.
a) Use of chloroform as a solvent causes slow accumulation of chlorine
which is a toxic substance and probably an auxin inactivator too.
b) Diethyl ether, if used as a solvent, bring about oxidation of the auxin in
the presence of a spontaneously formed peroxide. This can, however, be
avoided if the solvent is distilled with ferrous sulfate and calcium oxide
before use.
c) During auxin extraction, a new auxin may be produced which may thus
contaminate the auxin to be extracted. The difficulty can be overcome
by employing Gustafson’s technique which involves the boiling of plant
material for about a minute prior to extraction.

BIOASSAY OF AUXINS:
The term bioassay refers to determining the amount of active
substance present in the plant tissues. In the various methods employed for
bioassay the activity of the auxin is in general determined by making if available
in certain concentration to a seedling (or an ovary or a root) and the degree of
either acceleration or inhibition of growth is recorded.
One of the most commonly employed method of bioassay of auxins,
as devised by Went (1928), is known as Avena curvature test. The test involves
the following steps.

9
 Went found that the degree of angular curvature of coleoptile tip is
proportional, within limits, to the concentration of auxin present in the agar block.
This fact he made the basis of his Axena test.
Kogl and Haagen-Smit (1931) used Avena test as the unit of
measurement. It is termed Avena Eiaheit and abbreviated as A.E. One A.E. is
defined as the amount of auxin present in an agar block (2 x 2 x 1cm) which
produces a curvature of 100 to a decapitated Avena coleoptile when placed
eccentrically on it for 90 minutes.

BIOCHEMISTRY OF AUXINS:
Application of Avena test to a wide variety of substances led to the
discovery of auxins in human urine, Kogl and Haagen-Smit (1931) isolated 40 mg
of auxin (which they named as auxin a) from as much as 3.3 gallons of human
urine. Three years later, Kogl and his associates isolated two more compounds
with auxin activity. These were named as auxin b and heteroauxin and were
obtained from corn germ oil and human urine respectively.

1. Auxin a. auxentriolic acid :

It occurs at the meristematic apices (buds and growing leaves) both
in the free state and bound to plasma proteins. It is a weak acid and is soluble in
water, alcohol, ether and chloroform. It is stable in acid solutions but decomposes
in alkaline solutions (i.e. acid-stable and alkali-labile).

2. Auxin b auxenolonic acid:

It is present in corn germ oil. Other vegetable oils, malt and a fungus
called Rhizopus. It is also a weak acid and is soluble in water. Alcohol, ether and
chloroform. It is both acid labile and alkali-labile. This as well as auxin a both are
derivatives of cyclopentene.

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3. Heteroauxin, indole-3-acetic acid:
It is of universal occurrence in plant and is also synthesized by
microorganisms including certain bacteria, yeasts and fungi like Rhizopus. It is
resistant to alkalies whereas destroyed by acids an undergoes rapid decomposition
on heating. Unlike the first two it can be easily synthesized in the laboratory.
Chemically, it is a monobasic acid of a relatively simple structure.

NATURAL AUXINS
Besides the above-mentioned auxins, certain other compound have
recently been shown to occur in plants. These show similar behaviour in terms of
their effect on growth, although usually less intense. The principal naturally-
occurring auxins in plant that have been definitely identified, isolated, purified and
their chemical structure determined are all indole derivatives, (refer Fig. 32-5), as
mentioned below:
1. Indole-3-acetic acid, IAA
2. Indole-3- acetonitrile, IAN

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 3. Indole-3-acetaldehyde, IAC
4. Ethylindoleacetate
5. Indole-3-pyruvic acid, IPyA
6. Indole-3-ethanol, IEtOH

Controversy exists regarding whether ethylindoleacetate occurs

naturally or is produced during extraction as an artifact. The presence of indole-3-
ethanol is also not conclusively established.
Non-indole compounds, e.g., some fatty acids may also possess
auxin-like properties. But they have not yet been properly characterized.
The position of the side chain in the ring structure in indole-3-acetic
acid (IAA) appears to be highly specific for activity, since 1-,2- and 4- indole
acetic acids are only very slightly active in bioassay. Substitution with halogens,
like fluorine and chlorine can result however, in very active derivatives e.g. the 4-
chloro and 6-chloro compounds. The replacement of an aromatic CH of IAA by N
can give rise either to 7-aza (indole-3) acetic acid, a synthetic auxin for the
structure of this and other synthetic auxins) which is less active in the Avena test
or to another synthetic auxin, indazole-3-acetic acid which has activity equal to
IAA. Indole-3-acetonitrile (IAN) may be converted into either IAA or IAc.
Depending on whether C=N is replaced by COOH or CHO, respectively.
Replacement of the mino group (NH) in IAA by a S atom produces another
synthetic auxin called thianaphthen-3intoleacetic acid which is quite active in
various bioassay tests (Allsopp.1965) Indole -3-propionic acid (IPA) is less active
than IAA, but indole-3-butyric acid (IBA) is less active than IAA, but indole-3-
butyric acid (IBA) is more active than IPA, however in some tests as in case of
rooting of cuttings, IBA.

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Tests as in the case of rooting of cuttings, IBA is even as active as or more active
than IAA. It is noteworthy that the side chains having even number of C atoms are
more active than those having an odd number. It is hypothesized that the side
chain is oxidized to IAA before the compound shows a biological activity.

Indoleacetonitrile (IAN):
A nitrile derivative of IAA, is a neutral substance and has been
obtained in crystalline form from many plant materials. It is believed that the
nitrile derivative (i.e. IAN) as such is inactive and has to converted to IAA in
order to be active. IAN, upon alkali hydrolysis, yields IAA, IAN promotes growth
of only those plant organs (like Avena coleoptlie, bean seeds tomato ovary etc)
which possess the enzyme indoleacetonitrilase, while tissues (like pea roots)
which cannot convert IAN to IAA because of the absence of indoleacetonitrilase,
are unreactive to IAN. However, in higher concentrations, IAN is inhibitory like
IAA. In addition to acting as an auxin precursor. IAN is also known to be an
activator or booster of IAA responses.

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Ethylindoleacetate :
It is the ethyl ester derivative of IAA and has been isolated from
many plant tissues. Both IAN and ethylindoleacetate seem to be more active than
IAA. In those tissues which are capable of converting them into IAA. IAN,
ethylindoleacetate and indoleacetaldehyde, IAC (the aldehyde derivative of IAA).
All three exist in nature as auxin precursors and all of which can be converted into
IAA.

SYNTHETIC AUXINS :
Several small organic molecules have been synthesized which show
biologic properties characteristic of indole-3-acetic acid (IAA), though not in all
respects. They are usually derivatives of benzoic acid, indole-3-acetic acid or
naphthalene acetic acid. And acidic side chain. Unsaturation in the rung and an
unfilled ortho position usually characterize many active molecules, although no
general rule can be framed as some thiocarbamates. Which do not satisfy most of
the requirements are quite active. The D-isomer is more active than the L-isomer,
(cis-cinnamie acid) is an auxin whereas trans-cinnamic acid behaves as an
antiauxin. The 3-‘D’ structure of the molecule and the spatial relationship between
the side chain and the aromatic ring, fi present, determine biologic activity. Some
of the potent synthetic auxins are TIBA, 2-4-D, 2.3.5-T, NAA and NOA.
Phenylacetic acid (PAA) is, however, a week auxin.

14
Fig. Some synthetic auxins

15
BIOGENESIS (= SYNTHESIS) OF AUXINS :

Folke Skoog (1937) of the University of Wisconsin, for the first

time, experimentally proved that tryptohan is an auxin precursor in higher plant.
Since then a wide variety of plant tissues (like leaf, stem, buds, coleoptile, overy,
pollen, embryo, endosperm and callus tissue) have been shown to convert
tryptophan to indoleacetic acid (Larsen, 1951). In fact, it is quite probable that all
living plant tissues may have the capability of bringing about this conversion.
Although tryptophan is the primary precursor of IAA. About half of dozen indole
compounds have been found to serve as potential precursor of IAA.
For the enzymic production of IAA a plansible hypothesis has been
suggested by Wildman, Ferri and Bonner in 1946. According to this hypothesis
(Fig. 32-7), tryptophan, liberated by hydrolysis of proteins, undergoes either
oxidative amination first and then decarboxylation or vice versa to yield
indoleacetaldehyde (IAc). IAc is then oxidized to yield the free auxin
Indoleacetadehyde, thus, acts an intermediate metabolite as well as an immediate
precursor of IAA.
The capability of living plant tissues to form auxin from tryptophan
has been demonstrated for spinach (Wildman et al, 1947), pineapple (Gordon and
Nieva. 1949) and many others. The three essential conditions for IAA synthesis
are the presence of light, zinc and an enzyme system.

DISTRIBUTION OF AUXINS:
The greatest concentration of auxins is usually found in the growing
apices of the plant, i.e. in the coleoptile tip, in buds and in the growing tips of
leaves and roots. However, auxin is found widely distributed throughout the plant
body. In general, it may be stated that where there is active growth, there is auxin
production. The formation of auxin by mature organ like leaf, however, suggests
that growth may not be the pre-requisite to auxin production.
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 Thimann (1934) studied the distribution of auxin, in detail in
etiolated Avena coleoptile. He found that the concentration of auxin drops as one
progresses from the coleoptile tip to its base, the highest concentration being at the
tip and the lowest at the base. In one progresses further from the base of the
coleoptile along the root, there is stready increase in auxin contentill a maximum
is reached in the root tip. Of the two maximal values, that for the stem tip is much
higher than that for the root tip.
Thimann and Skoog (1934), while working on Vicia faba seedling
grown in light, found the concentration of auxins in various organs in the
following descending order
Apical buds > Young leaves > Mature leaves
The amount of diffusible auxin per hour for these organs was found
to be approximately in the ratio of 12:2:1.
Van Overbeek (1947) studied the distribution of both free and bound
auxins in pineapple. He found that large quantities of free auxin occurred in apical
buds and lowest amount in mature leaves. For bound auxin, however, the
condition was found to be reverse.
A few other examples of auxin concentration studies are given in
Table.
Table : Amount of auxin present in different organ of some plants
Sr. Maximum Concentration found
Plant Organ
No. ( in µg IAA equivalent)
01. Corn Endosperm 105,000
02. Lily Stem tip 83,900
03. Oat Grain 1,000
04. Rice Endosperm 250
05. Turimp Seed 250

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CONCENTRATION OF AUXINS :
The concentration of auxins, which has a profound influence on
growth changes, varies from organ to organ. The effect of auxin concentration on
the shoots is quite different from the optimum concentrations for growth
promotion were found to be between 10-11 and 10-9 for roots, 10-8 and 10-3 for
stems and 10-5 and 10-3 for floral buds. (After Leopld AC and Thimann KV.
1949) that on the roots. Higher concentration of auxin, which has growth-
stimulatory effect on the shoots, is growth-inhibitory for the roots, the later
growing better at much lower concentration. Thus in general, the optimum range
of concentration for elongation in stems is much higher than for the roots. The
stems grow best at an auxin concentration of 1.0 mg/ litre. Whereas the optimum
concentration for the roots is of 0.001 mg/ litre.

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TRANSLOCATION (=MOVEMENT OF AUXINS) :
The auxins are transported in plants from one organ to the other. The
usual direction of auxin transport is downward but when added to the soil these are
absorbed by the roots and carried upward along with transpiration stream to
various plant organs. The prevailing downward movement takes place through the
living phloem cells whereas the upward movement occurs through the dead xylem
elements.
The most striking characteristic of auxin movement is its almost
strict basipetal (from apex to base) polarity. This has been demonstrated in various
plant organs such as oat coleoptiles (Went and White, 1939), petioles of leaves
and herbaceous and woody stems (Oserkovsky. 1942). The actual proof of polar
transport was furnished by Went. He showed that if an agar block containing auxin
be affixed to the morphologically upper end of a coleoptile segment and a block of
pure agar to the lower end, auxin would move and collect in the agar block at the
lower end. But if the coleoptile segment is inverted, no translocation of auxin
would occur. The translocation of auxins in oat takes place through the
parenchyma tissue.
In Coleus, however, Leopold and Guernsey (1953) have shown that
the basipetal polarity becomes progressively weaker as the distance from the shoot
apex increases. Further, in the Jocobs (1961) has also shown that it Coleus stem
sections, the ratio of basipetal to acropetal transport of auxin is 3:1. in fact, there
exists a polarity gradient in Coleus from a complete basipetal polarity in
vegetative apex to a complete acropetal polarity in root apex with gradual
transition in between.
The velocity of auxin transport varies from 26 min per hour to 64
mm per hour (Rajgopal, 1967). These rates are higher than the rates of diffusion.
The velocity of auxin transport is unaffected by temperature, although the amount

19
of auxin transported ins proportional to the temperature. also the distance over
which the transport occurs does not influence the velocity.

MECHANISM OF AUXIN ACTION:

The action of auxins, like growth itself, seems to be a complex of
many functions. Although the mechanism of auxin action may, in part, be
attributed to each of these functions, none of these can account for in toto the
multifarious effects of these growth substances. The various views put forward to
explain auxin behaviour may be, for convenience, grouped under following five
headings.

1. Molecular reaction:
Skoog (1942) expressed the view that the auxin may act like a
coenzyme and serves as a point of attachment for some substrate onto an enzyme
regulating growth. The molecular configuration of the auxin affects the activity by
altering the fit and functioning of this molecular union. The higher auxin
concentrations would inhibit growth owing to separate molecules combining with
the enzyme and the substrate.
Muir et al. (1949) advanced the hypothesis that the auxins (exp.
Phenoxy acids) may combine with some material (e.g. protein ) in the cell at two
points, the ortho position of the ring and the acid group of the side chain.
Foster and his associates (2952) put forward a theory of auxin action
by 2-point attachment.
It is presumed that the enzyme is attached to a substrate to form an
enzyme substrate complex and that the complex may, the dissociate to produce the
end product of the reaction (i.e. growth) and regenerate the enzyme. Considering
the enzyme as the material with which auxin reacts. The theory may thus be
expressed as.
E+S = ES Growth + E
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 The inhibitory effect of high auxin concentration on growth may
well be explained by this hypothesis. This would result from two auxin molecules
becoming attached to the receptor substrate. One at each of the points of
attachment and each preventing the functioning of the other.

2. Enzymatic effects:
The fact that growing tissues, upon treatment with auxin show an
increased activity of a number of enzymes has proved to be the basis of these
theories. Northen (1942) pointed out that the auxin causes decrease in cytoplasmic
viscosity and also brings about dissociation of the cytoplasmic proteins. The latter
effect would result in an increase in water permeability and also in the osmotic
value of the cytoplasm. These effect ultimately lead to enhanced enzymic activity.
Burger and Avery (1942) demonstrated that some dehydrogenases,
under certain conditions, could be stimulated by auxin. Thimann (1951). However,

21
found that the auxins act as agents protecting certain growth enzymes from
destruction rather than as substances activating enzymes.

3. Osmotic effects:
During the process of growth the cell increases in volume due to
water uptake. The uptake of water occurs due to changes in the cytoplasm itself
(esp. changes in the osmotic value) or due to changes in permeability of the cell
wall and the cell membranes.
Czaja (1935). For the first time, stated that the auxin may increase
the volume of the cell which could result directly in water uptake and growth. Van
Overbeck (1944). However pointed out that growth is not necessarily associated
with an increase in osmotic value.
Commonor and his associates (1942-43) suggested that since water
uptake in growth may be linked with respiration, the process of growth may be
explained as due to the osmotic uptake of water which is, in its turn, activated by
respiratory uptake of salt.

4. Cell wall effects:

Hyen (1940) attributed growth to the dynamic function of the cell
wall instead of the cytoplasm. He observed that auxin application increases
flexibility and extensibility of the cells wall. This results in lowering the wall
pressure around the cell wall, thus permitting water uptake due to this simple drop
in turgor pressure.
The elasticity of cells always increase at the start of cell stretching
but decreases again before the cells have reached maturity. Thus, increasing
elasticity cannot cause elongation but is connected with the elongation process.
Ruge (1942) has suggested that cell elongation proceeds in two different phases:

22
(a) an increasing extensibility of the wall without synthesis of new all material.
(b) a hardening of wall with a deposition of new wall material through either
intussusception or apposition.

5. Toxic metabolism:
Besides promoting growth, the auxins can also inhibit elongation. In
short this inhibition is brought about by high auxin concentration whereas in roots
the inhibition is induced even by relatively low auxin concentrations. This
inhibition is thought to be due to excess auxin molecules inactivating the sites of
auxin action and thus, checking maximum growth response.
Van Overbeck (1951) proposed that growth regulator toxicity may
be a result of an alternation of metabolism in such a manner that unsaturated
lactones are accumulated in plant tissues. These are toxic to plants when applied
in higher concentrations. Such toxic compounds may accumulate in plant tissues
following application of such hormones as 2, 4-D.

23
PHYSIOLOGICAL ROLES OF AUXINS (EFFECTS OF AUXIN):
It was previously thought that the sole function of auxins was to
promote cell enlargement. But the work done in later years has proved them to be
deeply associated with a variety of functions. In some cases they act as
stimulating agent, in others as an inhibitory agent and in still others as a necessary
participant in the activity of other phytohormones such as gibberellins and
cytokinins. The various growth process in which the auxins (both natural and
synthetic) play their role are discussed below:

1. Cell elongation:
It is usually considered that cell elongation occurs only in the
presence of auxins and also that the rate of elongation is directly proportional to
the amount of auxin applied provided no other factors are limiting. But relatively
high concentrations usually exert inhibitory effect on this phase of growth.
Auxins also play a significant role in the elongation of petiole, mid
rib and major lateral veins of the leaves. Thus, adenine favours enlargement in
detached leaves of radish and pea. Similarly, coumarin has been shown to
promote expansion of leaves in some plants.
As osmotic equilibrium exists in a cell where the turgor pressure
developed is counterbalanced by the wall pressure acting in opposite direction.
Regarding the mechanism of cell elongation, it is thought auxins stimulate cell
elongation by modifying certain conditions responsible for this equilibrium
(Devlin, 1969). These modifications include:
(a) an increase in osmotic contents of the cell.
(b) an increase in permeability of the cell to water
(c) a decrease in wall pressure
(d) an increase in wall synthesis and
(e) an inducement of specific RNA and protein synthesis.

24
2. Cambial activity:
The trees exhibit growth by developing buds which later on open.
This then followed by elongation of the young stems. This resumption of growth
by cambial cells is activated by the auxins which move basipetally in the stems
from developing buds. Snow (1935) has shown that a steady supply of auxin a at
1/1,00,000 mg per hour (or of IAA at 1 / 500,000) mg per hour) from a gelatin
block, upon affixing it to the cut end of a decapitated shoot of sunflower
(Helianthus annuus) seedling, stimulated meristematic activity of the cambium.

3. Callus formation and galls:

Besides acting as stimulants of cell elongation, the auxins may also
activate cell division. This may be illustrated by applying 1% IAA in lanolin paste
to a debladed petiole of a bean plant. This causes prolific division of parenchyma
cells resulting in the formation of a swelling or callus tissue at a point where the
auxin in applied. The amount of callus tissue formed is directly proportional to the
concentration of IAA applied (Ropp, 1950).

4. Rooting of stem cuttings (= Formation of adventitious roots):

It is a common observation that the pressure of buds on a cutting
favours development of roots when the lower end is dipped in a suitable rooting
medium. Developing buds are effective in accelerating root formation. Young
leaves also favour the initiation of roots on the cuttings. These observations led to
the suggestion that the root formation is favoured by the auxins which are
synthesized in the buds and young leaves and are later translocated to the basal
part of the cutting.

25
5. Apical dominance:
As the apical bud is intact on the plant, the growth of the lateral buds
remains suppressed. Upon removal of the apical bud. The lateral bud nearest the
apical bud establishes its dominance over the remaining buds, causing them to
become inactive again. This inhibitory effect of a terminal bud upon the
development of the lateral buds is called apical dominance and produces a cone-
shaped plant.

6. Delay (or inhibition) of abscission of leaves:

The abscission of leaves can be delayed or inhibited by the
application of auxins on the surface of the lamina or on the cut surface of a
debladed petiole. The controlling behaviour of the auxins on the abscission was
first noted by Laibach(1933) who showed that the extract of orchid pollinia is
capable of preventing the leaf fall. Since then, enough work has been carried out in
this direction. Addicott and Lynch (1955) have proved conclusively the delaying
effect of IAA on the abscission of various plant organs.
As to the mechanism of abscission, it has been suggested that the
leaf fall is retarded by the basipetal migration of a hormone from the blade to the
base of the periole. Removal of the leaf blade eliminates the supply of hormone to
the abscission zone and thus induces leaf fall.

7. Flowering:
A flowering hormone, florigen, is produced in the leaves under
correct light and dark period. It moves first down the petiole and then up the stem
to the growing apex where it causes the development of floral buds in place of
vegetative buds (Cajlachjan, 1936).

26
8. Fruiting :
Auxins play significant role in fruiting by modifying it in one of the
following ways:
A. Fruit Setting:
Fruit set refers to the changes in the ovary leading to the
development of the fruit. These changes are usually induced after pollination and
fertilization.

B. Fruit thinning:
In many instances, the trees bear extensively large number of fruits.
This causes the trees to fail to produce average number of new flower buds. Such
trees, therefore, have to produce fruits either a alternate years (alternate bearing) or
if yearly, the number of fruits is greatly reduced (infrequent bearing). These trees,
obviously, require thinning.

C. Control of premature fruit dropping:

In many fruit trees, the unripe fruits fall of on account of the
formation of an abscission layer, thus causing serious losses in yield. This problem
has now been successfully overcome in many cases like apples by the application
of auxins which prevent the formation of abscission layer and thus check
preharvest drop of the fruits.

D. Improving fruit quality:

The various processes like colouration, softening, sweetning and
ripening are all involved in improving the quality of the fruit.

27
E. Increase in respiration:
James Bonner (1953), for the first time, recognized that auxins
stimulate the process of respiration. And as such a direct relation between growth
due to auxin treatment and the rate of respiration has been found. Greater the
growth, higher is the rate of respiration.

F. Increased resistance to frost damage:

In parsnip, the tops resist damage by frost on treatment with 2, 4, 5-
T. Similarly, the application of 2, 4, 5-T in apricot fruits before the onset of frost
resulted in less damage than the untreated fruits.

G. Great weapon of war:

Auxins when applied in greater concentrations on enemy crop fields
by air may cause devastation of land and thus form the basis of what is called
biological warfare.

Biosynthetic Pathway of Indole-3-acetuc acid:

IAA is an important regulator of plant growth, the source of this
compound in plant remains surprisingly elusive, with advent of more sophisticate
& sensitive analytical techniques, the tang held notion of tryptophan as
predominant precursor is being challenged considering that plant cannot survive
without IAA & inability to generate mutants completely deficient in auxin (Klee &
Estell 1991). It is reasonable to extent that several IAA. Biosynthetic pathway
exists, even within a single plant the relative activities of which depend on plant
species & on the development state of the plant.
The indole-3-acetic acid is produced by most of the bacteria that
inhabit the rhizosphere of plants & roots nodules of plants. Several different IAA
biosynthesis pathways are used by these bacteria with a single bacterial strains

28
sometimes containing more than one Pathway. The level of expression of IAA
depends on the biosynthesis on chromosomal or plasmid DNA and their regulatory
sequences; and the presence of enzymes that can convert active, free IAA into an
inactive, conjugated from. The role of bacterial IAA in the stimulation of plant
growth and phyto pathogenesis is considered. In culture the symbiotic isolated
from the nodules, produced high amount of IAA, when tryptophan was supplied in
the medium as a precursor. The symbiont preferred L-isomer over the D-or-L-
isomer of the tryptophan IAA production.
Several Trp-dependent pathways, which are generally named after
an intermediate, have been proposed; the tryptamine pathway and the Indole-3-
acetaldoxime (IAQX) pathway.
The IPA pathway (Trp-IPA- indole-3-acetaldehyde (IAA/d) - IAA]
is important in some IAA-Synthesizing micro-organism (Koga 1995) and may
operate in plants as well (Coohey and Nonhebel, 1991). IPA is found in
arabidopsis seedlings (Tam & Normanly, 1988) but genes encoding a Trp to IPA
or an IPA decarboxylase that converts IPA to IAA / Id have not been identified in
plant. The final enzyme in the proposed IPA pathway is an IAA Id specific
aldehyde oxidase protein (AA01) that has increased activity in the IAA
overproducing mutant (Seo-et al. 1988).
The IAM pathway (Trp-IAM-IAA) is a second microbial pathway
that also may act in plants. In Agrobacterium tumifacians & pseudeomnas
syringae for e.g. Trp mono-oxygenase (IoaM0 convert IAM to IAA (patten and
Cylick, 1996).
The indole-3-acetic acid pathway involves the transmutation of
tryptophan to indole-3-pyruvic acid followed by Indole-3-pyruvic acid followed
by decorboxylation to indole-3-acetaldehyde & further oxidation to IAA.

29
 Tryptophan may also converted to indole-3-acetaldehyde via indole-
3-acetaldehydoxime (Luowig Maller & Hilgenberg; 1988, Rajagopal et al… 1993)
or through tryptamine.
The common final step in the synthesis of IAA from
indoleacetadehyde can occure via on aldehyde oxidase or an aldehyde
dehydrogenase (tsurusaki et al. 1997). The intermediate indole ocetaldoxime can
also be converted to IAA via indole-3-acetonitrile (Ludwing muller & Hilgenberg;
1990)
Activites of Tryptophan monooxigenase & indoleacetamide
hydrolase that catalyses the two step synthesis of IAA from tryptophan in the
indole-3-acetamide pathway have been identified.

30
31
OVERVIEW OF GENUS RHZOBIUM:
Rhizobium Gram negative rods ranging from 0.5-0.9 x 1.2 – 3.0 mm,
commonly pleomorphic under adverse growth conditions, usually contain granules
of poly- β- hydroxybuterate which are refractile by phase contrast microscopy,
non spore forming. Motile by one polar or subpolar flagellum or two to six
pertitrichous flagella fimbriae have been described on a few strains these are
aerobic & possessing a respiratory type of metabolism with oxygen as the terminal
electron acceptor. It is able to grow well under oxygen tension less than 1.0 kpa
optimum pH for growth is 6-7 and optimum temperature is 25-30 0C. Colonies are
circular, convex, semi translucent, raised and mucilaginous usually 2-4 mm in
diameter in 3-5 days on yeast extract mannitol mineral salt agar, pronounced
turbidity develops after 2-3 dasy in agitated broth. It is chemo-organotrophic
utilizing, a wide range of carbohydrates and salts of organic acids as a carbon
sources without gas formation, cellulose and starch are not utilized. It produces an
acidic reaction in mineral salt medium containing mannitol or other carbohydrates.
Growth on carbohydrate media is usually accompanied by copious extracellular
slime. Ammonium salt, nitrate, nitrite and most amino acid can serves as nitrogen
sources. Some strains will grow in a sample. Mineral salts medium with vitamin
free case in hydrolysate as the sole source of both carbon & nitrogen peptone is
poorly utilized & casein and agar are not hydrolysed by this organism some
strains require biotin or other water soluble vitamin.
The organisms are characteristically able to invade the root hairs of
temperate zone and some tropical zone leguminous plants (family leguminaceae)
and insite production of root nodules where in the bacteria occur as intracellular
symbionts. All strains exhibit hast range affinities (hast specificity). The bacteria
present in root nodules are in pleomorphic forms (bacteroids) which are normally
involved in fixing atmospheric nitrogen into a combined form Ammonia)
utilizable by host plant. The mol. % G+C of the DNA is 59-64 ™.

32
 The nitrogen requirement can be satisfied by nitrate and by many
amino acid e.g. short chains peptides, certain amino acids e.g. glycine may be
inhibitory. There have been several reports of possible nitrogen fixation by Free
living strains but the situation in this respect is not yet clear, some strains like
(R. meliloti) in particular anaerobically produce N2 from nitrate.
Most strains lack the ability to absorb congo red form a yeast extract
mannitol mineral – salts medium containing a 0.0025% final concentration of this
dye. The temperature range which is highly strain dependant, is 4-420C however,
growth at 40C is rare, and only R. meliloti can grow at 42.50C & the temperature
maximum for R. leguminasarum is 380C. The pH range for this genus is 4.5-9.3,
R. meliloti being the most alkali-tolerant species. There are weekly proteolytic, but
most strains produce a slow digestion in litmus milk, forming an upper clear
“serum zone” usually with a slightly alkaline reaction or no change. R.melioti
strains tend to produce on acidic reaction.
Members of genus are common soil inhabitants. Identification is
relatively easy of isolated from host plant nodules but different if isolated from the
soil or if a non infective mutant. The ability to cause nodule formation
(infectiveness) is more stable than the ability to fix nitrogen in symbiosis.
(effectiveness). Strains tend to lose effectiveness after serial cultivation on media
containing certain amino acids, especially O-forms, and after long term storage of
continuous growth on laboratory media. Infectiveness and effectiveness represent
discrete phenomenon and vary within wide limits, depending upon genetic factors
in both bacterial strains & host plants.
There are three main species of genus Rhizobium are as.
1) Rhizobium leguminosarum.
2) Rhizobium meliloti
3) Rhizobium loti

33
 There are three biovars of Rhizobium leguminosarum are found as
trifoli, phaseoli and viceae.

* Nitrogen Fixation by Rhizobium:

The process of conversion or reduction of molecular nitrogen in the
atmosphere to the nitrogenous compounds such as NH3 is called as “Nitrogen
Fixation”
Number of micro-organism are able to use the molecular nitrogen in
the atmosphere which is reduced to nitrogenous compounds. Nitrogen fixation is
of two types
1. Symbiotic type
2. Non symbiotic type

1. Symbiotic N2 fixation :
The organism fixes nitrogen by carving symbiotic association with
leguminous plant. i.e. the organism Rhizobium Bradirhizobium, Sinorhizobium,
MesoRhizobium, Azorhizobium are gram-ve motile rods, infection of roots of
leguminous plants with appropriate species of these genus for e.g. Rhizobium leads
to the formation of root nodules that are able to convert gaseous nitrogen to
combined nitrogen i.e. N2 to NH3 a process is called as symbiotic N2 fixation.

2) Non symbiotic N2 fixation:

In this process microorganisms independently fix the nitrogen while
growing in soil. The best example of nonsymbiotic nitrogen fixing organism is
Azatobacter.

34
The Rhizobium-legume symbiosis:
The rhizobia re soil microorganism that can interact with leguminous
plant to form root nodules within which conditions are favourable for bacterial
nitrogen fixation. Legumes allow the development of very large rhizobial
populations in the vicinity of their roots. Infection and nodule formation require
the specific recognition of host and Rhizobium, probably mediated by plant lectin.
Penetration of the host by a compatible nodule, and a process of differentiation by
both partners then ensures. In most cases the rhizobia alter morphologically to
form bacterioids, which are usually larger than free-linving bacteria and have
altered cell walls. At all stages during infection, the bacteria are bounded by host
cell plasmalemma. The enzyme nitrogenase is synthesized by the bacteria and, if
legheamoglobin is present, nitrogen fixation will occur. Legheamoglobin is a
product of the symbiotic interaction, since the globin is produced by the plant
while the haem is synthesized by the bacteria. In the intracellular habitat the
bacteria are dependent upon the plant for supplies of energy and the bacteroids, in
particular, appear to differentiate so that they are no longer able to utilize the
nitrogen that they fix. Regulation of the supply of carbohydrate and the use of the
fixed nitrogen thus appear to be largely governed by the host.
The interaction between species of Rhizobium and leguminous plants
which results in the formation of nitrogen fixing root nodules requires the
expression of genes of both partners in a manner that is not well understood.

* Indole acetic acid production by Rhizobium:

Besides being a N2 supplier to plants through symbiotic association
i.e. symbiotic N2 fixation in the plants of family leguminaceae, one of the most
important ways that bacteria affect growth and development is by producing
Indole-3-acetic acid (IAA) that this hormone lead to plant root system
development & subsequently nutritional uptake increase by plant. Many of

35
rhizobium species able to produce IAA. Indole-acetic acid is one of the most
physiologically active auxin.s Rhizobium use L-Tryptophan as a precursor for the
production of IAA. IAA is the common product of L-Tryptophan metabolism by
several other bacteria. It also secretes other hormones i.e. plant growth regulators
(auxins, gibberllines & ehtylenes siderophores, HCN and antibiotic production.
Rhizobium synthesize the Indole-3-acetic acid & provide it to the
plant & thus contribute to plant’s endogenous pool of auxin.
Genes for the enzyme involved in indole acetic acid production are
found on plasmid as well as on bacterial genome, although it is easy to measure.
The concentration of IAA produced by bacteria in test tube by Salkowski reagent
test, it is difficult to predict the level that will be produced in the root nodules of
plants as expression of these genes is controlled by many as yet poorly understood
genetic & environmental factor.

Historical Background:
1. Ahmad F. Ahmad S. Khain MS. (2005) Indole acetic acid production by
Azatobactor & pseudomonas in presence & absence of L-TRP.
2. A. Ernstsen, G. Sandberg, A, A crozier and C.T. Wheeler, Endogenous
indoles and the biosynthesis & metabolism of indole-3-acetic acid in
cultures of Rhizobium phaseoli.
3. Asana, Mani & Prakash (1995) worked on “Effect of Auxin & Yield of
wheat.”
4. Aloni R. (1995) Induction of vascular tissues by auxin and cytokinin.
5. Avery G.S. (1933) differential distribution of phytohormones in the
developing leaf of nicotine.
6. Balas N. Wong LM, Ke. M. Theologis, Two auxin responsive domains
interact positively to induce expression of the early indoleacetic acid
inducible genes.

36
7. Burger and Avery (1942) demonstrated some dehydrogenases, under certain
conditions stimulated by auxin.
8. Charles Dorwin (1960) power of auxin movements in plants.
9. Cheryl L. Patten and Bernard R. Click (1996) Bacterial Biosynthesis of
Indole-3-acetic acid.
10. Costacurta a., Vandesley Den (1996) worked on synthesis of
phytohormones by plant associated bacteria.
11. Estelle M. Polor (1998) auxin transport.
12. Hafiz Naeem Asghar, Zahir Ahmad Zahir, Abdul Khatiq & Mahammad
Arshad (2000) Isolation of Rhizobacteria from different Rapeseed varieties
& their potential for indole-acetic-acid production.
13. Julius Von Sachs (1880) organ forming substance in plants.
14. Kawaguchi M, Syonok (1996) the excessive production of indole-3-acetic
acid & it’s significance in studies of biosynthesis of this regular of plant
growth & developed.
15. Kogl and Haagen – Smit (1931) Isolated auxin from human urine.
16. Leopod & Fuente (1968) auxin transportation in plant cells.
17. Manutis S., Shafrir, H. Epstein E. Lichter A., arash I, (1994) Biosynthesis
of Indole-3-acetic acid via the indole S. acetamide pathway in streptomyces
spp.
18. Peter Boysen & Jenson (1910). The material nature of hormones.
19. Prist W. Went (1928) isolation, extraction and bioassay of growth
promoting substances.
20. Soding (1925) decapitation of coleoptile markedly retards the rate of cell
division.
21. Starling (1902); worked on plant hormones.
22. Thimann (1948) designated the term phytohoromones.

37
23. Went, 1926; Kogl & Haagen-Smit (1931) IAA is the first description of
polor auxin transport.
24. Went (1928) Avena curvature test.

38
 MATERIAL & METHODS

* Glasswares :
All glasswares procured from store of microbiology department
D.S.C.L. of different companies such as “Qualigens & Borocil”. These were
washed with double distilled water and HNO3 .
Petriplates,
Test tubes
Conical Flasks (250 ML, 100 ML & 50 ML)
Screw Cap tubes
Beakers
Pipettes etc.

* Laboratory equipments:
- Incubator
- Hot air over.
- Digital spectrophotometer
- Centrifuge machine.
- pH meter
- Refrigerator,
- Shaker

Sterilents & Disinfectants :

- Alcohol.
- Dettol

- Root nodules of gram (cicer aerietinum) (collected from Udgir, M.I.D.C.,

area)

39
 Media used (All media procured from store of microbiology
department, D.S.C.L. of different companies such as ‘Himedia’, ‘Qualigens’,
‘Loba-chemi’).

1) Congo Red Yeast extract mannitol Broth:

Composition-In distilled water g/l : mannitol, 10.0; K2HPO4, 0.5; MgSO4-
7 H2O, 0.2; Nacl, 0.1; Yeast extract, 1.0; Congo red solution, 1%, 2.5 ml.

Preparation:
Sterilize at 1210C for 20 min. congo red solution sterilized separately and
added in the medium before pouring in the petriplates. For agar preparation
add 2.5% agar in broth medium before sterilization.

2) L-Tryptophan
3) Peptone Water:
Composition in distilled water g/l : Peptone, 20; sodium chloride (Nacl),
5.0; pH 7.2.

Preparation:
Dissolve all the components are in warm water, pH is adjusted and
autoclave at 1210C for 15 minutes.

4) Sugar Fermentation medium:

Composition:
Peptone, 100 ml; Test Sugar, 0.5 to 1.0 g; Bromothymol blue, 0.25 ml.
Preparation:

40
 Sugar is added in peptone water adjust the pH, distribute in tubes, sterilize
in autoclave at 1210C for 20 minutes (steam sterilize at 1000C for 1 hr. if
the sugar is thermolabile like diasaccharide lactose)

5) H2S production medium:

- Peptone water.
- Lead acetate
- Filter paper strip (Lead acetate paper strip)
6) Congo red yeast extract mannitol broth having pH 5.0, 8.0, 9.0 & 9.5.

7) Congo red yeast extract mannitol broth containing 2% Nacl.

8) Gram Staining Kit
- Crystal Violet
- Gram iodine
- Alcohol
- Safranin
9) Chemicals: (Analytical test – Salkowski Reagent test)
- O-phosphoric acid.
- Salkowski reagent

Composition:
Ferric Chloride (FeCl3) - 0.5 m
Perchloric acid - 85%

Preparation :
1 ml of 0.5 FeCl3 Solution added to 30 ml of 33% perchloric acid, the
resulting solution is nothing but salkowski reagent.
10) Standard Indole-3-Acetic Acid (Powdary form)

41
Methods:
[A] Enrichment of Rhizobium :
- For enrichment purpose healthy root nodules with a small portion of root
attached are placed in tube & washed thoroughly with tap water & then
with distilled water to remove contaminants & adhering soil particles.
- Then immersed in 0.1% acidified HgCl2 for 5 minutes.
- Then treated with 10 ml of 95% ethanol for 2-3 minutes.
- Then again these nodules were washed with tap water & blot dried with
blotting paper.
- Aseptically crushed the nodules with glass rod or dissected by using
nichrome blade.
- 1% of suspension prepared in saline was inoculated into YEM Broth and
incubated at 300C + 20C for 48 hrs.

B] Isolation of Rhizobium:
- For Isolation, a loopful of pellicle was streaked on Yeast extract mannitol
agar plates.
The plates were kept for incubation at 300C + 20C for 48 hrs.
- After incubation the colony characteristics & gram nature of isolated
colonies were noted down.

C] Identification of Rhizobium species:

For identification, the colonies isolated were studied for Biochemical
characteristics like sugar fermentation H2S, production, growth in presence
of 2% Nacl, Growth at different pH was studied.
The purity of culture was checked. The slants were then used as
stock culture for indole acetic acid production.
BIOCHEMICAL TESTS: PERFORMED:
42
1) Sugar Fermentation Test:
- Peptone water is prepared & pH is adjusted distributed in tubes as 10 ml in
each tubes.
- Tubes were labeled for different sugars as glucose, galactose, fructose,
Arabinose, Xylose, maltose, Sucrose, Lactose, mannitol etc.
- 1% each sugar was added in their respective tubes containing peptone
water.
- Durhams tubes were added in all tubes for detection of gas production.
- Then after sterilization 0.1 ml of bacterial suspension was inoculated into
each tube : & incubated at room temp. i.e. 300 + 20C.
- After incubation observed for colour change (i.e. acid production) & gas
production.
- Results were noted down.

2) H2S production Test:

- Peptone water was prepared in test tube into that 0.1 ml of suspension was
inoculated.
- The lead acetate paper strip was instead upwardly in hanging position.
- The tube was incubated for 24 hrs & observed for blackening of lead
acetate paper strip.

3) Growth in presence of 2% Nacl:

- Yeast extract mannitol broth was prepared & 2% Nacl was added into that
& sterilized.
- Then 0.1 ml of suspension was added in the test tube containing 10 ml of
YEMB medium having 2% Nacl observed for growth after incubation of 24
hrs.
4) Growth at pH 5.0, 8.0, 9.0 & 9.5 :

43
- Congo red yeast extract mannitol broth medium was prepared & distributed
in test tubes as 10 ml in each tube.
- Tubes were labeled as pH 5.0, 8.0, 9.0 & 9.5 respectively./
- Then pH was adjusted by using 0.1 N NaOH & 0.1 NHCl solution.
- The medium was sterilized at 1210C for 15 minutes.
- 0.1 ml of suspension was inoculated in each tube.
- After incubation observed for growth (turbidity)

D] Screening of bacterial isolates for Indole-3-acetic acid production:

The test strain of Rhizobium was screened for indole acetic
acid production. The test bacterial culture was inoculated in respective
medium (congo red yeast extract mannitol broth) with tryptophan (1 mg /
ml) and one was without tryptophan and incubated at 300C + 20C for 48 hrs.
cultures were centrifuged at 3000 r.p.m. for 30 min. supernant was taken
and production of IAA was tested by Salkowski Reagent Test.

E] Detection of Indole-acetic acid production:

Salkowski reagent test was used for detection of Indole acetic acid
produced by micro-organism. This test was described by Sowar
et al.(1992). In this test, 5 ml of culture broth was taken & centrifuged at
3000 r.p.m. for 30 minutes To the 2 ml of supernatant 2 drops of
orthophosphoric acid was added with well shaking after it 4 ml of
Salkowski reagent with constant shaking was added. Observed for the
development of pink color which indicates the IAA production. O.D. was
read at 530 nm and the level of IAA was estimated by standard graph of
IAA.

44
F] Optimization of Tryptophan concentration for Indole acetic acid
production:
- The optimization was carried out to determine tryptophan concentration at
which Rhizobium gives high yield of Indole acetic acid.
- For this purpose the suspension of or pure culture of Rhizobium was
inoculated in 10 different flasks of cango red yeast extract mannitol broth
having different concentrations of tryptophan ranging from 100 µg /ml to
1000 µg /ml.
- After incubation Indole-acetic acid produced was determined by Salkowski
reagent test, by taking supernatant from centrifugation.

G] Production of Indole-acetic acid by Rhizobium:

- 500 ml of congo red yeast extract mannitol broth medium was prepared in
1000 ml flask to this tryptophan was added at the final concentration of
700 µ g /ml.
The medium was sterilized at 1210C for 20 minutes, cooled &
inoculated with Rhizobium.
- The flask was incubated at 300C for 148 hrs. at 300C + 20C. Samples were
withdrawn every 24 hr. and growth of IAA concentration determined.
- After 148 hrs of incubation the time duration within at which maximum
IAA production was determined graphically by observing O.D. at 530 nm
every after 24 hrs.

H] Extraction of IAA:
- For extraction of crude IAA from culture broth. Bacterial cells were
separated from supernatant by centrifugation at 10,000 r.p.m. for 30 min.
- The supernatant was acidified to pH 2.5 to 3.0 with 1 N HCl and extracted
twice with ethyl acetate at double the volume of the supernatant.

45
- Extracted ethyl acetate fraction was evaporated to dryness in a china dish at
400C in boiling water bath. Then the extract was dissolved in methanol and
stored or kept at – 200C (in refrigerator).

I] Effect of crude (extracted) IAA on root elongation:

- In petriplate the filter paper was placed by cutting appropriate size of
petriplate.
- It was moistened with distilled water.
- The four to five seeds of cicer aerietinum was surface sterilized & soaked
in water for 2-3 hrs then kept in petridish on the weted filter paper.
- Then like this control & Std. plate & test plates were mode.
- Three plates were there, one for extracted crude auxin, second for standard
IAA Solution & third is of distilled water treatment.
- To the Test Plate 1-2 drops of extracted auxin was dropped on seeds.
- To the standard plate 1-2 drops of standard auxin solution was dropped on
seeds.
- To the control plate no treatment of auxin was given to the seeds i.e. only
distilled water was supplied for germination.
- These plates were incubated.
- Daily the plates were treated with their related solutions upto 5 days &
observed for germination.
- The length of root was measured in each plate.

46
* Observation and Results:
1) Isolation of Rhizobium species on Congo red yeast extract mannitol
agar at 300 + 20C for 24 hrs.

Colony Characters:
Size Shape Colour Morgin Elevation Surface Opacity Consistency
Pale
2 mm Circular Regular Elevated Smooth Opoque Moist
white

Gram Nature Motility

Gram negative rods Motile

Result :
The Rhizobium spp. was isolated on congo red yeast extract
mannitol agar plates.

47
Biochemical Characterization of Rhizobium Species
Sugar Fermentation Results
Glucose +
Galactose +
Fructose +
Arabinase +
Maltose +
Sucrose +
Lactose +
Mannitol +

Growth at pH Result
5 +
8.0 +
9 +
9.5 -

Growth at 39-400C +
Growth in Presence
Of 2% Nacl +
H2S Produced +

2) Identification of Rhizobium Species:

Result :
From colony characters, Gram Staining, motility and Biochemical
characterization (tests) the isolated organism, identified was Rhizobium melioti.

48
* STANDARD GRAPH OF IAA :
Stock solution of IAA was prepared having 1000 µml. i.e. 100 mg
of standard IAA is dissolved 100 ml of Sterile D/W. From the Stock the dilutions
are prepared form 100 µml. to 1000 µml.

Concentration Optical
Sr. Stock Distilled
of IAA ( µ density at 530
No. Solution (ml) Water (ml)
g/ml) nm
1 0.1 0.9 100 0.540
2 0.2 0.8 200 0.700
3 0.3 0.7 300 0.780
4 0.4 0.6 400 0.935
5 0.5 0.5 500 1.335
6 0.6 0.4 600 1.586
7 10.7 0.3 700 1.840
8 0.8 0.2 800 1.900
9 0.9 0.1 900 1.960
10 1 0 1000 1.985

49
* Effect of different concentration of L-Tryptophan on production of
IAA :
The production of IAA at each tryptophan concentration i.e. 100 µ
ml. to 1000 µml. was determined by Salkowski reagent test & optical
density was observed at 530 nm, it gives the amount of Indole-acetic-acid
produced at each tryptophan concentration.

* Observation Table:
Tryptophan Amount of IAA
Sr. Optical density at
concentration ( µ produced ( µg/
No. 530 nm
g/ml) ml)
1 100 0.816 310
2 200 0.669 250
3 300 0.438 170
4 400 0.717 270
5 500 1.066 400
6 600 1.092 410
7 700 9.105 420
8 800 0.975 370
9 900 1.018 380
10 1000 0.965 360

Result :
The maximum concentration of Indole-3-acetic acid produce was
found at the 700 µg/ml concentration of Tryptophan.

50
* Effect of Incubation period on production of IAA:
The organism Rhizobium melioti inoculated into YEM broth having
700 µg/ml of L-TRP and incubated at 300 + 20C for 148 hrs. At each 24 hrs IAA
production was checked by Salkowski reagent test & O.D. was observed at 530
nm concentration of IAA produced was determined.

* Observation Table:
Concentration of
Sr. Inculbation Time Optical density at
IAA produced µ
No. in hours 530 nm
g/ml
1 24 0.515 200
2 48 0.780 300
3 72 1.200 450
4 96 1.018 380
5 120 0.970 370
6 148 0.680 250

Result :

The maximum IAA production was found at 72 hrs of incubation

period. Firstly it increases upto 72 hrs then in declines slowly.

51
 DISCUSSION

Indole-3-acetic acid is the most physiologically active & principle

form of auxin. It generate majority of auxin effects in plant such as cell division,
cell enlargement, shoot & root growth, & vascular tissue differentiation in plants.
Thus it is agriculturally important product & plays important role in plants.

Thus the project work “Production of Indole-3-acetic acid (auxin) by

Rhizobium. Species isolated from root nodules of Cicer aerietienum and its effect
on the germination of seeds.” Has been undertaken to determine the indole acetic
acid production ability of Rhizobium melioti and the effect of extracted auxin on
the germination of seeds.

The Rhizobium spp. was grown on conge red yeast extract mannitol
broth medium provided with Tryptophan and incubated for IAA production. Then
by using Salkowski regent test the production of IAA is checked by observing the
development of pink color & O.D. was observed at 530 nm optimization of
Tryptophan concentration was carried out to detect the optimum concentration of
Tryptophan at which higher amount of IAA production takes place & it is
demonstrated.

Based on morphological, cultural & biochemical characteristics, the

root nodule isolates of cicer aerietinum were identified as Rhizobium melitoti.
The Identification was done according to Bergey’s manual of determinative
bacteriology (dordan, 1984). The IAA production by Rhizobium started after 24 hr
& reached at a maximum after 72 hr. when the bacteria reached a stationary phase
of growth & then decreased slowly. This could be due to better utilization of
medium components for IAA production by this isolate.

52
 The bacteria i.e. Rhizobium preferred L-Tryptophan for maximum
IAA production. The effect of different concentrations of L-Tryptophan revealed
that maximum growth and IAA production was observed at 700 µg / ml. of
Tryptophan concentration. It indicates that Rhizobium utilizes the L-Tryptophan as
the main precursor component of the IAA metabolism pathway.

The effect of IAA produced by Rhizobium evaluated at 700 µg / ml.

concentration of Tryptophan on the germination of cicer aerietinum (i.e. root
elongation was highest with IAA extracted from Rhizobium spp. As compared to
control & standard. Some seed showed no germination because it may be due to
inhibition of growth due to higher concentration of IAA.

The effect of auxin on plant seedlings are concentration dependant

i.e. Low concentration may stimulate growth while high concentration may be
inhibitory. Different plant seedling respond differently to variable auxin
concentrations & type of micro-organisms.

The findings of the present investigations highlighted that IAA

producing bacteria from local soil & nodules of different plants of family fabaceae
could be easily isolated & may be exploited after strain improvement for local use.

53
 CONCLUSION

From this study it is clear that Rhizobium isolates from root nodules
of cicer aerietinum is able to produce Indole-3-acetic-acid (auxin) by using L-
Tryptophan as a main precursor of IAA metabolic pathway. The highest IAA
metabolic pathway at 700 µg/ml. of L-Tryptophat & al 72 hrs of incubation.
There fore the symbiont was responsible for higher IAA content of root nodule
(i.e. growth & development of cells of tissues)

54
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