Algal Research 7 (2015) 100105

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Algal Research
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In vitro regeneration of Arachis hypogaea L. and Moringa oleifera Lam.

using extracellular phytohormones from Aphanothece sp. MBDU 515
Manickam Gayathri a,1, Pichaimuthu Senthil Kumar b,1,
Azhagiya Manavalan Lakshmi Prabha b, Gangatharan Muralitharan a,
a
Department of Microbiology, Centre of Excellence in Life Sciences, Bharathidasan University, Palkalaiperur, Tiruchirappalli 620 024, Tamilnadu, India
b
Department of Plant Sciences, Centre of Excellence in Life Sciences, Bharathidasan University, Palkalaiperur, Tiruchirappalli 620 024, Tamilnadu, India

a r t i c l e i n f o a b s t r a c t

Article history: The present study aims to develop an efcient and simple protocol for in vitro plant regeneration using
Received 9 July 2014 cyanobacterial liquid medium. The ability of the unicellular cyanobacterium Aphanothece sp. MBDU 515 to pro-
Received in revised form 7 November 2014 duce the phytohormone indole-3-acetic acid (IAA) in the medium was demonstrated and chemically analyzed
Accepted 17 December 2014
by Salkowski assay, High performance liquid chromatography (HPLC) and Gas chromatographyMass spectrom-
Available online xxxx
etry (GC/MS). The effect of cyanobacterial extracellular product (CEP) for in vitro propagation was evaluated on
Keywords:
two economically important plants Arachis hypogaea and Moringa oleifera. The effect of commercial phytohor-
Aphanothece mones 6-Benzylaminopurine (BAP) and Indole-3-butyric acid (IBA) in shoot and root induction, respectively
Arachis hypogaea was compared with CEP in both the plants. In the present study, it was observed that 1 mL of CEP per 10 mL of
Cyanobacteria MS basal medium showed an increase in % of shoot and root responses, shoot and root lengths and average num-
Moringa oleifera ber of roots per shoot compared to the control (MS basal medium). These effects of CEP are comparable to the
Phytohormones commercial phytohormones BAP and IBA, tested. The tested CEP markedly reduced the accumulation of phe-
Plant tissue culture nolics in A. hypogaea explants. Our study suggests that CEP may be used for micropropagtion at industrial scale
instead of commercially available phytohormones.
2014 Elsevier B.V. All rights reserved.

1. Introduction secondary metabolites [7]. There are many examples of cyanobacteria

Efcient in vitro regeneration of plants depends upon reliable tissue
culture techniques, which can bring about successful application of bio-
technology in crop improvement [1]. Currently this technology has been
successfully applied to a wide range of crops, forest and fruit, with com-
mercial micropropagation laboratories worldwide. However, the tech-
nology is capital, labor and energy intensive. Hence, it is necessary to
have low cost options for tissue-culture technology. In low cost technol-
ogy, cost reduction is achieved by improving process efciency and bet-
ter utilization of naturally available resources in place of commercial
chemicals [2]. Two classes of phytohormones i.e., cytokinins and auxins
are very important for in vitro regeneration of plants [3]. It is already
well evident that cyanobacteria are competent in both accumulating
and releasing a variety of phytohormones such as auxins, gibberellins,
cytokinins, vitamins, polypeptides, and amino acids, which promote
plant growth and development [4,5].
Cyanobacteria are among the most geographically widespread group
of organisms known, and even dominate some aquatic and terrestrial en-
vironments [6]. They are the most prolic source of wide variety of
Corresponding author.
E-mail address: drgm@bdu.ac.in (G. Muralitharan).
1
Equally contributed.
from different genera that produce both exogenous and endogenous
IAAs [3,810]. In vitro regeneration of Oryza sativa, Triticum aestivum, So-
lanum tuberosum, Brassica oleracea, Nicotiana tabacum and the ornamen-
tal plant Lilium alexandrae were successfully accomplished in the MS
medium supplemented with cyanobacterial extracts [3,1115]. Prasanna
et al. [14,16] screened the rhizosphere soil of twelve rice cultivars from
six different regions of India and reported that Anabaena strains CW1
and RP9 exhibited promising IAA production. Interestingly, the Anabaena
strain CW1 was isolated from the rice eld of Aduthurai, Tamilnadu,
India. However, only few species of unicellular cyanobacteria have
been tested so far for the production of phytohormones.
Hence this study aims to determine the efciency of unicellular
cyanobacterial strain Aphanothece sp. MBDU 515 isolated from the rice
eld of Thanjavur district for its plant growth promoting potential in
Arachis hypogaea and Moringa oleifera. Peanut (Arachis hypogaea) is
one of the principle economic oilseed legumes and is largely cultivated
in many tropical and subtropical regions of the world and they are very
recalcitrant to tissue culture regeneration. Therefore, many efforts have
been devoted to develop an efcient in vitro regeneration system [17].
The few reports on the tissue culture of M. oleifera described clonal
propagation through the use of nodal explants taken from non-
specic sources, either from young seedlings or mature plants [18,19].
The preservation of the Moringa species is thus of great concern from

http://dx.doi.org/10.1016/j.algal.2014.12.009
2211-9264/ 2014 Elsevier B.V. All rights reserved.
 M. Gayathri et al. / Algal Research 7 (2015) 100105
biodiversity, ethnobotanical, dietary and pharmacological perspectives
[20].
2. Materials and methods
2.1. Cyanobacterial strain and growth conditions
The free living Aphanothece sp. MBDU 515 was isolated from the rice
elds of Thanjavur district, Tamilnadu and was maintained in axenic
form in the germplasm of Department of Microbiology, Bharathidasan
University, Tiruchirappalli. The organism was grown in a 250 mL Erlen-
meyer ask containing 100 mL BG 11 medium and incubated at 25 C
under a light-ux density of 50 mol/m2/s for 15 days [21].
2.2. Preparation of cyanobacterial extracellular product (CEP)
The CEP was prepared by harvesting 15 days old culture of the tested
cyanobacterium at 10,000 rpm for 15 min at 4 C and supernatant was
collected. The supernatant was sterilized by ltration before being
used at various concentrations along with basal Murashige and Skoog
(MS) medium [12].
2.3. Extraction and identication of IAA
The cyanobacterial culture broth was centrifuged at 10,000 rpm for
15 min at 4 C and the supernatant was collected. The 40 mL of superna-
tant was extracted with 80 mL of ethyl acetate in separating funnel. Ethyl
acetate fraction was recovered and evaporated. The residue was dis-
solved in methanol and used for further analysis. HPLC equipped with
a constant ow pump (Shimadzu, Japan) was used. Separation was ac-
complished under reversed phase isocratic conditions with (Shim-Pack
CLC-Octadecyl silane) ODS-C18 column (4.6 mm ID 25 cm) and guard
column (Shim-Pack G ODS) (4 mm ID 1 cm) and mobile phases of
100% methanol. The ow rate was adjusted to 1 mL/min for analysis
and a UV absorbance at 254 nm was used as a detector. Prior to GC/MS
analysis, samples were resuspended in an appropriate volume of metha-
nol. Samples of 1 L in methanol were injected into the GC/MS equipped
with HP5 fused silica capillary column (30 m 0.25 mm 0.25 m).
Helium was used as a carrier gas at a ow rate of 1 mL/min and operated
in a split less mode. Injector and detector temperatures were both set at
220 C. The oven temperature was held at 80 C for 2 min, then ramped
at a rate of 20 C min1 and then nally programmed from 220 C to
250 C at a rate of 10 C min1 for 10 min. MS was performed with an
AGILENT Gas chromatograph coupled to a GCmate II Joel (Japan)
ion trap mass spectrometer using electron ionization (EI) in a
positive mode with ionization energy of 70 eV. Spectra were
recorded from m/z 100 to 2500 and with a scan rate of 0.5 s scan 1.
2.4. Plant material and culture media
Healthy and nodal explants of Arachis hypogaea and Moringa oleifera
were collected from 45 days old plants grown under green house condi-
tion at Bharathidasan University, Tiruchirappalli, Tamilnadu, India.
Shoot tip, internode and nodal explants were washed thoroughly in
running tap water for 20 min, before being dipped in 70% ethanol for
60 s and rinsed with sterile distilled water and surface sterilized using
0.1% mercuric chloride solution then again washed well with sterile dis-
tilled water (3 to 5 min each). Apical and nodal regions were used for
the following experimental setup.
2.5. Phytostimulation experimental setup
To assess the effect of CEP on shoot regeneration, the explants were
subcultured into fresh culture tubes containing MS basal medium (MS),
MS + BAP (1.02.0 mg/L) and MS + CEP (0.51.0 mL of CEP in 10 mL of
MS basal medium, (hereafter 0.5 mL/10 mL and 1.0 mL/10 mL,
101
respectively) and allowed to grow for 15 days. The culture tube contain-
ing MS medium without the addition of plant growth regulators was
served as control. After 15 days, the explants from each experiment
mentioned above were transferred to new culture tubes containing con-
trol, MS + IBA (12.0 mg/L) and MS + CEP (0.51.0 mL/10 mL) for root
regeneration. For each treatment ten replicates were used. All the treat-
ments were repeated at least three independent times. The treated ex-
plants were maintained in a growth chamber at 25 2 C and
16 hour photoperiod and 8 hour dark period with PPFD of
50 mol m2/s provided by cool white uorescent lamps for 15 to
30 days to obtain shoot and root development [22]. The number of ex-
plants responded to shoot development (% of shoot response) was cal-
culated for each treatment. Also, the length of shoot responded was
measured, averaged and compared between control and treatments.
Similar measurements were carried out to calculate the percentage of
root response, the number of roots per explant and the length of roots.
2.6. Salkowski assay
The screening for the presence of IAA-like compounds was carried
out as described earlier [23]. The Salkowski reagent (1 mL of 0.5 M
FeCl3, 50 mL of 35% HClO4) [24] was added to the culture supernatant
in a ratio of 1:1 (v/v). The concentration of IAA in the culture medium
was determined by comparison with the standard curve. Auxin-like
substances were estimated by OD at 535 nm after 30 min in the dark
at room temperature. Salkowski assay was used for the determinations
of released, Salkowski-positive indolic compounds, particularly auxins.
The presence of IAA in the CEP was further conrmed using HPLC and
GC/MS analyses.
2.7. Statistical analysis
All experiments were conducted with ten replicates per treatment
and the mean values were compared using Duncan's Multiple Range
Test (P b 0.05) using SPSS v.20 for windows 7 Basic.
3. Results and discussion
In this study, MS medium was complemented with two different
concentrations (1 and 2 mg/L) of BAP and IBA, for shoot and root induc-
tion respectively and were compared with that of CEP activity. McKently
et al. [25] reported that the MS medium supplemented with BAP
showed maximum shoot regeneration from the explants taken from
the seed of peanut. In Arachis hypogaea, the MS medium supplemented
with 0.5 mL and 1 mL/10 mL of CEP showed a two fold increase in the %
of shoot response compared to the control. However, % of shoot re-
sponse between the MS medium supplemented with BAP and MS sup-
plemented with CEP at both the concentrations tested was statistically
similar. In addition, the shoot length was increased two fold in the MS
medium supplemented with CEP (1 mL/10 mL) when compared to
the MS medium supplemented with both concentrations of BAP, where-
as the shoot length in the MS medium supplemented with CEP (0.5 mL/
mL) was found to be statistically similar (Table 1a and Fig. 1a). Although
there is an increase in % of shoot response in BAP treated explants, the
shoot length is decreased when compared to control (MS basal medi-
um) in both cases (Table 1a and Fig. 1a). This is due to the accumulation
of phenolics (Fig. 2b) during shoot development. The absence of pheno-
lics might be attributed to the two fold increase in shoot length in CEP
treated explants (Table 1a).
For root induction in Arachis hypogaea, % of root response, average
number of roots/shoot and the root length were measured for each of
the treatments (Table 1b). The MS medium supplemented with CEP
(0.5 mL and 1 mL/10 mL) showed similar % of root response and average
number of roots produced per shoot compared to IBA at both concentra-
tions (1 and 2 mg/L), whereas, the MS medium supplemented with CEP
at 1 mL/10 mL and 0.5 mL/10 mL concentrations showed a three-fold
102 M. Gayathri et al. / Algal Research 7 (2015) 100105

Table 1
Effect of different concentrations of CEP on shoot response in Arachis hypogaea L. com-
pared to the commercial phytohormones BAP and IBA. (a) Shoot response and shoot
length in Arachis hypogaea L. (b) Root response, average number of roots per shoot and
root length in Arachis hypogaea L.

Treatments % of shoot response Shoot length (cm)

MS basal (Control) 40d 4b

MS + BAP (1 mg/L) 70c 3b
MS + BAP (2 mg/L) 80b 2.5b
MS + CEP (0.5 mL/10 mL) 80b 4b
MS + CEP (1 mL/10 mL) 90a 6a

MSMurashige and Skoog; CEPcyanobacterial extracellular product;

BAP6-Benzylaminopurine; values in the table represents mean (n = 10).
Mean having the same letters in column does not differ signicantly at P b 0.05
(Duncan's Multiple Range Test).

Treatments % of root Average no. of Root length

response roots/shoot (cm)

MS basal (Control) 0 0 0
MS + IBA (1 mg/L) 75d 1c 3c
MS + IBA (2 mg/L) 90b 3a 7b
MS + CEP (0.5 mL/L) 85c 2b 6b
MS + CEP (1 mL/10 mL) 92a 3a 9a

MSMurashige and Skoog; CEPcyanobacterial extracellular product; IBAIndole-3-bu-

tyric acid; values in the table represents mean (n = 10). Mean having the same letters
in column does not differ signicantly at P b 0.05 (Duncan's Multiple Range Test).

and two-fold increase in root length compared to the MS medium sup-

plemented with IBA (1 mg/L), respectively (Table 1b and Fig. 1b). This
clearly demonstrated the similar effects of CEP and IBA in root induction
of Arachis hypogaea. Our results agree with those obtained by

Fig. 2. The photographs showing the organogenesis of Arachis hypogaea L. (a) MS medium
(control), (b) shoot development in the MS medium + BAP (2.0 mg/L), (c) root develop-
ment in the MS medium + IBA (2 mg/L), (d) shoot development in MS CEP (1 mL/10 mL),
and (e) root development in MS + CEP (1 mL/10 mL).

extracellular products of cyanobacterium Phormidium subincrustatum

Fig. 1. Effect of different concentrations of CEP on Arachis hypogaea L. compared to the com-
mercial phytohormones BAP and IBA. (a) Shoot response in Arachis hypogaea L. (b) Root re-
sponse in Arachis hypogaea L. (Values indicated the root length and average no. of root
response). Means having the same letters do not different signicantly at P b 0.05 (Duncan's
Multiple Range Test).
on the regeneration of Wedelia trilobata L [26].
Browning and blackening due to excessive accumulation of pheno-
lics are the major drawbacks of in vitro culture of many economically
important plants. Interestingly, we found the absence of phenolic secre-
tion in shoot and root induction during CEP treatment in Arachis
hypogaea (Fig. 2d and e). When cells are damaged, the contents of cyto-
plasm and vacuoles are mixed and phenolic compounds can readily be-
come oxidized by air, which may inhibit enzyme activity and result in
the darkening of the culture medium and subsequent lethal browning
of explants or causes rooting deciencies. The oxidized phenols (poly-
phenol oxidase) are negatively correlated that produce highly toxic qui-
nones and polymerized material causing discoloration of the medium
and death of the cultured explants [27]. Notably, the CEP used in our
study also inhibited the accumulation of phenolics compared to that
of control treatment (Fig. 2a and b).
Another economically important plant Moringa oleifera was also
tested for root and shoot development with CEP. In Moringa oleifera,
the MS medium supplemented with 0.5 mL and 1 mL/10 mL of CEP
showed a similar level of % of shoot response as that of control and MS
medium supplemented with BAP in 1 mg/L and 2 mg/L concentrations,
whereas the length of the shoot in medium supplemented with both
BAP (Fig. 4b) and CEP (Fig. 4d) at both concentrations was signicantly
increased three fold when compared to control shoots (Table 2a, Figs. 3a
and 4a). Our results indicate that although CEP does not play any role in
% of shoot response, it has a prominent function in the regeneration of
shoot in Moringa oleifera. Similar effects were observed in % of root
 M. Gayathri et al. / Algal Research 7 (2015) 100105 103

Table 2
Effect of different concentrations of CEP on shoot response in Moringa oleifera Lam. com-
pared to the commercial phytohormones BAP and IBA. (a) Shoot response and shoot
length in Moringa oleifera Lam. (b) Root response, average number of roots per shoot
and root length in Moringa oleifera Lam.

Treatments % shoot response Shoot length (cm)

MS basal (control) 70e 2c

MS + BAP (1 mg/L) 75d 5a
MS + BAP (2 mg/L) 80c 6a
MS + CEP (0.5 mL/L) 82b 5a
MS + CEP (1 mL/10 mL) 90a 6a

MSMurashige and Skoog; CEPcyanobacterial extracellular product;

BAP6-Benzylaminopurine; values in the table represents mean (n = 10).
Mean having the same letters in column does not differ signicantly at P b 0.05
(Duncan's Multiple Range Test).

Treatments % of root response No. of roots Root length (cm)

MS basal (control) 0 0 0
MS + IBA (1 mg/L) 80d 4c 6c
MS + IBA (2 mg/L) 92b 6ab 8ab
MS + CEP (0.5 mL/L) 90c 5bc 7bc
MS + CEP (1 mL/10 mL) 95a 7a 9a

MSMurashige and Skoog; CEPcyanobacterial extracellular product; IBAIndole-3-bu-

tyric acid; values in the table represents mean (n = 10). Mean having the same letters
in column does not differ signicantly at P b 0.05(Duncan's Multiple Range Test).

response, number of roots formed and the root length of CEP (Fig. 4e)
and IBA treated explants (Table 2b, Figs. 3b and 4c).
The capacity of IAA-like production by the tested cyanobacteria was
determined using the Salkowski reagent. The extra-cellular ltrate of

Fig. 4. The photograph showing the organogenesis of Moringa oleifera Lam. (a) MS medium
(control), (b) shoot development in the MS medium + BAP (2.0 mg/L), (c) root development
in the MS medium + IBA (2 mg/L), (d) shoot development in MS + CEP (1 mL/10 mL), and
(e) root development in MS + CEP (1 mL/10 mL).

Aphanothece sp. MBDU 515 showed a positive color reaction indicative

Fig. 3. Effect of different concentrations of CEP on Moringa oleifera Lam. compared to the
commercial phytohormones, BAP and IBA. (a) Shoot response in Moringa oleifera Lam.
(b) Root response in Moringa oleifera Lam. (Values indicated the root length and average
no. of root response). Means having the same letters do not different signicantly at
P b 0.05 (Duncan's Multiple Range Test).
of a release of an IAA-like substance. About 8 g of IAA per mL of culture
medium was shown to be produced by the tested cyanobacterial strain.
The Salkowski method represents a fast and simple colorimetric assay
and was used already for the determination of IAA from various
cyanobacteria [23]. In cyanobacteria, the production of IAA is indepen-
dent of morphological complexity and intra- or extra-cellular symbiosis.
Out of the three unicellular cyanobacterial strains reported, only
Gloeothece PCC 6909 showed a positive Salkowski assay and produced
both exogenous and endogenous IAA [23].
To chemically authenticate the presence of IAA in the tested CEP,
HPLC and GC/MS analyses were performed. The HPLC elution prole
of the standard IAA showed a major peak at retention time of
3.01 min (Fig. 5a), while the elution prole of CEP conrmed the pres-
ence of IAA with peak at retention time of 2.89 min (Fig. 5b). The
mass spectrum of the tested cyanobacteria is shown in Fig. 5c. The IAA
molecule was observed by the precursor ion at m/z 175.2 and product
ion at m/z 129.6. Although other uncharacterized peaks are present in
the GC/MS spectrum, the morphogenetic effects of the tested plants
were due to the presence of IAA at m/z 175.2 and m/z 129.6 in the CEP
and this is in line with the retention time of standard IAA with CEP by
HPLC analysis. The GC/MS method was shown to be consistent for the
determination of both exogenous and endogenous IAA irrespective of
the strain examined and the time tested [23]. Similar characteristic
mass spectrum was shown using LC/ESI-ITMS and UPLCESIMS/MS
104 M. Gayathri et al. / Algal Research 7 (2015) 100105
Fig. 5. The identication of IAA in (CEP) prepared from Aphanothece sp. MBDU 515 by HPLC elution prole of (a) standard IAA, (b) CEP and (c) GC/MS analysis of CEP.
methods for the determination of IAA from plants and cyanobacteria,
respectively [8,28].
During the past decades, complex nutritive mixtures have been
added to plant tissue culture medium. Now-a-days media containing
chemically-dened compounds are commonly used. The in vitro cul-
tures of recalcitrant and economically important plants always need
other organic growth substances in addition to the plant hormones
[29]. In this study we have evaluated the plant growth promoting ef-
fects of CEP derived from axenic cultures of Aphanothece sp. MBDU
515 and showed the presence of IAA. The supernatant from cultured
cyanobacteria could be used as an alternative supplement for improved
shoot and root regeneration than the commercial hormones and also al-
leviate the barriers in micropropagation caused by synthetic chemicals
[30]. Our data suggested that Aphanothece sp. MBDU 515 grown medi-
um may be employed as an organic growth promoter instead of com-
mercially available one. Moreover cyanobacteria based IAA is more
advantageous that they inhibited the accumulation of phenolics, a
major setback in commercial phytohormone based tissue culture
technology. Further research is required to study the mode of action
and synthesis of IAA like compounds in cyanobacteria and also to deter-
mine the presence of the IAA homologues including indole-3 propionic
acid and indole-3 butyric acid.
Acknowledgments
The authors are thankful to Sophisticated Analytical Instruments
Facility of IIT, Madras for the analytical support. MG acknowledges
Bharathidasan University authorities for the University Research
Fellowship URF (05441/URF/K7/2013 dated 04.07.2013).
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