Exogenous application of tryptophan and indole acetic acid (IAA) to induce root nodule formation and increase yield of soybean.

Agricultural Science Research Journal Vol. 2(4) pp. 134 – 139, April 2012
Available online http://www.resjournals.com/ARJ
ISSN-L: 2026-6073 ©2012 International Research Journals

Full Length Research paper

Exogenous application of tryptophan and indole acetic
acid (IAA) to induce root nodule formation and increase
yield of soybean
Sudadi
th

Department of Soil Science, Faculty of Agriculture, Sebelas Maret University, Ir. Sutami road 36 A Kentingan,
Surakarta, Central Java, Indonesia 57126.
Author Email: sudadi_uns@yahoo.com
Abstract

This study aimed at understanding to what extent extracellular tryptophan and indole acetic acid (IAA)
induce soybean root nodules formation. Greenhouse experiment was conducted in plastic pots and
sterile sand planting medium, arranged in completely randomized design (CRD) with three replications.
The factors evaluated were, type of phytohormones (tryptophan and IAA), application time (early

planting, vegetative phase 1, vegetative phase 3) and hormone concentrations (0; 0.001; 0.01; 0.1; 1, 0
and 10, 0 ppm). The results showed that both tryptophan and IAA induced root nodules formation.
Additionally, the addition of extracellular hormones also increased shoot and root dry weight as well as
soybean yields. Highest root nodules number and soybean yield were taken from the treatment of 1.0
ppm tryptophan applied in early planting. It seemed that higher concentrations of tryptophan or IAA are
required when applied at early planting, presumably because of its concentration decreases prior to
root hairs formed.
Key words: extracellular IAA tryptophan, root nodules, soybean.
INTRODUCTION
The existence and effectiveness of root nodules is very
important for soybean plants. Effective root nodules are
able to meet up to 75% of the plant’s nitrogen
requirement (Jutono, 1985; Ohyama et al., 2009).
According to Ohyama et al. (2009), one reason for the
low soybean yields compared to its potential are the less
effective root nodule. The formation of root nodule is
affected by internal factors of the plant and bacteria,
including the ability of plant to excrete tryptophan and the
capability of bacteria to oxidize tryptophan into IAA (Gray
and Williams, 1971; Spaepen, 2007). Phytohormones

IAA (auxin) play an important role in early infection
process of root nodules (Gray and William, 1971;
Spaepen and Vanderleyden, 2010; Verma et al., 2010).
Infection process and development of root nodules were
induced by auxin
(Libbenga et al. cit. Mishra et al.,
1999; Spaepen et al., 2007; Spaepen and Vanderleyden,

2010).
In the normal process, Rhizobium oxidizes the amino
acid tryptophan excreted by soybean into IAA. There
were some possibilities, due to certain factors, such
environmental stress, including acidic pH, osmotic and
matrix stress, and carbon limitation or the present of plant
extracts or specific compounds (Spaepen et al., 2007),
the plants maybe unable to excrete the amino acid
tryptophan, so it does not form IAA and the formation
processes of root nodules are inhibited. Bhattacharyya
(2006) said that heavy metals such as Hg, Pb, Cd and Ba
+2

affect IAA production of Rhizobium with Hg has the
most inhibitory effect. These problems can be overcome
if both compounds can be provided in the form of
extracellular compounds. The aim of the research was to
study whether extracellular amino acid tryptophan and
IAA induced the formation of root nodules in soybean

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Figure 1. Effect of the type, application time and concentrations of extracellular phytohormones (amino acid
tryptophan and IAA) on the number root of nodules of soybean planted on sterile sand planting medium

plants and increased soybean yield.

obtained were analyzed statistically by F test at 5% level
of confidence, followed by Duncan's multiple range tests
in case of significant effects.

Materials and methods
The experiment was conducted in a greenhouse, using 1
gallon plastic pots with sterile sand planting medium.
Local soybean varieties and rhizobia inoculums from Soil
and Environmental Microbiology Laboratory, Department
of Microbiology, Faculty of Agriculture, Gadjah Mada
University, Yogyakarta, Indonesia were used. The
experiments were arranged in factorial Completely
Randomized Design (CRD) with three factor treatments:
i) type of extra cellular phytohormones (amino acids
tryptophan = F1 and IAA = F2), ii) application time (early
planting = V0; vegetative phase 1 = V1 and vegetative
phase 3 = V3) and iii) phytohormone concentrations (0;
0.001; 0.01, 0.1; 1, 0 and 10.0 ppm). Each treatment
combination was repeated three times. Three soybean
seeds were planted in each pot, and thinned to one plant
per pot a week after planting. Observed variables
included the number of root nodules, shoot and root dry
weight and plant yield. Sampling for observation of root
nodules and plant dry weight was carried on the growth

phase R1 (beginning flowering) while for plant yield at R8
(full mature pods) (Hidayat, 1985; Kamal, 2000). Nitrogen
fertilizer was applied at a dose of 20 kg N/ha before
planting and 40 kg/ha during pod filling (Afza, et al.,
1987), while P (75 kg/ha P2O5 ), K (100 kg/ha K2O)
(Pasaribu, Suprapto, 1985), and Ca and Mg (200 kg/ha)
(Jutono, 1985) were given prior to planting. Micro
nutrients were added in the following dosages (Ismunadji,
Mahmud, 1985): 10 kg/ha Fe, 10 kg/ha Mn, 4 kg/ha Zn,
10 kg/ha Cu, 0.2 kg/ha Mo and 1.2 kg/ha B. The data

Results and discussion
There is an opportunity to utilize extracellular
phytohormones to enhance root nodules formation.
Mishra et al., (1999) stated that the used of external
phytohormones showed a response in the formation of
root nodules. Application of exogenous IAA enhanced
nodulation on Medicago and Phaseolus vulgaris when
added at very low concentrations (up to 10-8 M) to the
medium (van Noorden et al. cit. Spaepen et al., 2007).

Extracellular auxin or naphtilic acetic acid (NAA)
stimulated formation and growth of Medicago sativa root
nodules and Arachis hypogaea
(Srinivasan and
Gopalkrisna cit. Mishra et al. 1999), while 1-napthoxy
acetic acid and
(p-chlorophenoxy)-isobutyric acid
increased root nodules number in nuts (Cartwright cit.
Mishra et al. 1999). Kamal (2000) stated that addition of
epi-brasinoloid on soybean growth media increased root
nodules number per plant. The formation of root nodules
could also be induced by other extracellular
phytohormones such as cytokinin (Nandwal, Bharti cit.
Mishra et al. 1999) and GA (Bishnoi, Krishnamoorty cit.
Mishra et al. 1999). According to my results, the analysis of
variance indicated that the number of soybean root nodules
was significantly influenced (P=0.000) by the type,
application time and concentration of extracellular
phytohormones (tryptophan and IAA). These effects are
depicted in Figure 1.

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Figure. 2. Effect of the type, application time and concentrations of extracellular
phytohormones (amino acid tryptophan and IAA) on root dry weight of soybean planted on
sterile sand planting medium.

The number of soybean root nodules were influenced
significantly (P = 0.000) by the type, application time and
concentration of extracellular phytohormones (tryptophan
and IAA). The extracellular addition of tryptophan
resulted in higher number of root nodules than IAA.
Presumably, this because tryptophan was a precursor of
IAA, which was required in root nodules formation
processes. Spaepen et al. (2010) stated that application
of exogenous tryptophan increased strongly IAA
production in various bacteria. IAA was involved in many
processes of nodule formation by rhizobia in legume

plants such as founder cell specification, nodule initiation
and differentiation, nodule number and nitrogen fixation.
Many researchers showed that tryptophan played a key
role as precursor of IAA biosynthesis in various bacteria
(Datta, Basu, 1997; Ghosh, Basu, 1997; Datta, Basu,
1998; Datta, Basu, 2000; Celenza, 2001; Asghar et al.,
2002; Bhattacharyya, 2006; Ghosh, Basu, 2006;
Spaepen, Vanderleyden, 2010; Chaiharn Lumyong,
2011). Patten and Glick (2002) showed that IAA
accumulates in the culture medium of Pseudomonas
putida GR12-2 only when grown in the presence of
exogenous tryptophan. Tryptophan applied to the soil will
be oxidized to IAA, which then stimulated root growth and
induced nodules formation. On the other hand, IAA
applied into the soil, maybe metabolized or decomposed
that decreased its concentration and so its influence on
root nodules formation processes. Yang, Saleh (1975)
said that IAA was rapidly destroyed in the presence of
2+
Mn , oxygen and sulfite ion. According to Gregory (2006)

root nodules formation was initiated by cortical or
pericycle cell divisions and their differentiation are
controlled by various hormones, especially auxin and
cytokinin. Baca, Elmerich (2007), Spaepen and
Vanderleyden (2010) and Verma et al. (2010) stated that

plant hormones including auxin (IAA) play a key role in
various physiological processes during plant growth and
development. Higher root nodules number were obtained
from the treatment combination of (F1 V3 0.001 ppm) as
well as (F1 V0 1.0 ppm) and (F1 V0 10.0 ppm). In addition,
there was a possibility that the higher influence of
tryptophan than IAA was due to its influence on root
growth, as shown in Figure 2. Verma et al. (2010) stated
that the influence of PGPR on the stimulation of
nodulation was most commonly an indirect effect by
stimulated root growth, which provided more sites for
infection and nodulation. Reed et al. (1998) stated that
inhibition of auxin transport from shoots to roots inhibits
lateral root development thus reducing the number of root

nodules per plant because some of soybean root nodules
are on it lateral roots.
The effect of tryptophan in root dry weight was more
prominent than that of IAA. This is presumably, because
amino acid tryptophan is oxidized by rhizobium to IAA
which stimulates root growth of soybean plants.
Bandurski, Nonhebel (1983), Pandey, Sinha (1996) and
Guilfoyle et al., 1998) stated that at cellular level, auxin
affects cell division, elongation and differentiation,
whereas at plant level auxin affects plant tropism, plant
morphology, shoots dominance, leaves senescence, leaf
abscission, flowering, fruit formation and maturation, and
root growth.
Effect of the type, application time and the
concentration of extracellular amino acid tryptophan and
IAA on soybean seeds dry weight showed a similar trend
to its influence on root nodules number and root dry
weight, namely that the effect of tryptophan was higher
than of IAA, as presented in Figure 4. Seeds dry weight
was influenced significantly (P = 0.0002) by the

interaction of the type, application time and concentration

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Figure 3. Nodules formed at lateral roots of soybean planted on sterile
sand planting medium treated with amino acid tryptophan at vegetative
(V) growth stage 1 (V1 T0.001 ppm) and V3 (V3 T0.1 ppm).

Figure 4. Effect of the type, application time and concentrations of extra-cellular
phytohormones (amino acid tryptophan and IAA) on seed dry weight of soybean
planted on sterile sand planting medium.

of these extracellular phytohormones (tryptophan and
IAA). Highest soybean grain yield (0.711 g / plant) was
obtained from the treatment combination of amino acid
tryptophan (F1) applied at vegetative growth stage 3 (V3)
with a concentration of 1.0 ppm. This, because
extracellular application of the tryptophan at V3 stimulates
root growth and root nodule formation higher than at V0 or
V1, presumably because at V3 the plant root had grown
more extensively and provided more sites for infection
and nodulation, so it stimulates higher on N2 fixation and
grain yield. The concentration applied into the soil was
1.0 ppm, to ensure that there was enough tryptophan to
produce IAA.

Conclusions and suggestions
The extracellular application of amino acids tryptophan
and indole acetic acid (IAA) induce root nodule formation,
as indicated by the increased of root nodules number,
increase root dry weight and soybean yields planted on
sterile sand planting medium. Amino acid tryptophan
increased number of soybean root nodules higher than
did IAA. This, presumably, tryptophan played a key role
as precursor in IAA biosynthesis. When it was applied
into the soil, it would be oxidized by rhizobia to IAA which
in turn stimulated the processes of root nodules
formation. While for the IAA, when it was applied into the

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soil, maybe it concentration declined immediately,
because of damage or metabolized to another
substances before the root hairs were formed, so its
influence on the root nodules formation was smaller.
Higher root nodules number were obtained from the
treatment combination of (F1 V3 0.001 ppm) as well as (F1
V0 1.0 ppm) and (F1 V0 10.0 ppm). Highest soybean grain
yield (0.711 g/plant) was obtained from the treatment
combination of amino acid tryptophan (F1) applied at
vegetative growth stage 3 (V3) and concentration of 1.0
ppm. More researches are needed to find out local and
cheap sources of amino acid and IAA and application
field methods that enhance the highest root nodules
formation, N2 fixation, and plant growth and yields of
soybean, groundnut, pea, and others leguminous plants.
Acknowledgement
The author would like to thank to The Director of
Research and Community Services, the Directorate
General of Higher Education, Ministry of National
Education of Indonesia for financial support of the
Fundamental research from which this article was written.
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