INTERNATIONAL JOURNAL OF SCIENCE SCHOLARS, Vol-1, issue-03, pp.

117-128, 2017
Advance access publication- 15 July, 2017
This paper is available online free of all access charges

ISSN NO.- 2456-8929

INTERNATIONAL JOURNAL OF SCIENCE SCHOLARS

The House of the Written Word

RESEARCH PAPER

EVALUATION OF EFFICACY OF BIOTIC STRESS ON PRODUCTION OF

BIOACTIVES FROM IN VITRO CULTURES OF KENAF.

Manisha1*, Navjot2*

1. Punjabi university, Patiala

2. Punjabi university, Patiala

Received- 28 June 2017; Revised 10 July 2017; Accepted- 15 July 2017

Abstract-

Leafy vegetable are important for consumers in view of their nutritional and nutraceutical
potential which is mainly due to the presence of several bioactive compounds viz.,
flavonoids, phenolics, vitamins etc. Kenaf belongs to Malvaceae in mainly known throughout
world for its best fibres, but in some parts of the world the leaves are considered edible and
also its seed oil. Under this context, an attempt has been made to investigate selective
bioactives in leaves and in vitro cultures of kenaf (Hibiscus sabdarifa), and also to find out
the efficacy of some biotic stress factor on production of phenolics, flavonoids and ascorbic
acid. Though the levels of these bioactive in vitro established normal root cultures was less
compared to that of leaves of ex vitro grown plant, these established root cultures would be
having implications in using them as a model for pursuing the biosynthetic pathways of
respective bioactives. The biotic elicitor stress under in vitro conditions showed significant
enrichment of TPC and ascorbic acid content but at the same time there was no increase in
case of total flavonoids content. As plants under field conditions are exposed to various biotic
and abiotic stress factors certainly there will be significant changes in bioactives profile in
leaves of kenaf. The data generated in the present study would suggest pursuing similar

117 Corresponding Author- Manisha| Copyright, IJSS, 2017

studies under ex vitro conditions for better understanding the efficacy of biotic elicitors for
nutritional improvement of kenaf.

Keywords- Nutraceutical, Bioactives, Kenaf, flavonoids, phenolics, ascorbic acid.

Introduction-

Secondary metabolites produced by plant extracts can be used for pharmaceutical,

nutraceutical and biological studies. Phyto-constituents especially pigment and phenolic
compounds function as antioxidants to minimize oxidative damage of living cells and to
protect oxidative deterioration of food. There is always a growing interest and necessity to
identify the new biomolecules with promising activity from plants. Discovering such new
properties in a range of natural biological resources will lead to new ingredients and products,
and new opportunities for food processing industry. Apart from the nutritional significance,
some plant metabolites known as phytochemicals have also been used for health food
applications. In general both fruits and vegetables are known to be a good source of
phytonutrients. Leafy vegetables are also important as affordable source of nutraceuticals in
food applications. Lesser known edible plant species that are used traditionally in different
parts of our country and are sporadically used for edible purposes by local communities needs
to be pursued on large scale levels to promote for food applications and to alleviate
malnutrition problem. Hibiscus cannabinus (Kenaf) contains many nutritional and
nutraceutical components such as antioxidant molecules, beta carotene, iron, folic acid and
dietary fibres. The high content of iron in leaves would allow it to be used as a nutritional
supplement in diverse food products that would answer anaemic problems in humans. Since
H. cannabinus is an important green leafy vegetable, there is a lot of scope on studies of
nutritional enhancement through biotechnological interventions. More so presence of acidic
flavour helps in the preparation of various food formulants for value addition. Through
extentive studies have shown the presence of various nutraceutical components in green leafy
vegetables (e.g., antioxidants and prebiotics), not much of information is available on H.
cannabinus as a whole. Nutritional enhancement can be prepared for value addition. The
sustainability of the plant in general is not a problem since enough quantity will be available
as it grows wild in various places of southern India. In order to understand some of the
important biomolecules biosynthesis pathway it is necessary to have some in vitro models.
Once, identifying the key step of respective metabolic pathway, there is a great possibility of
augmenting the targeted molecule either through biotechnological methods or alternate
environmentally friendly methods, so that it could be possible to achieve enhanced levels of
bio-actives for value addition in Kenaf. Under this context, it is necessary to utilize the
elicitor mediated technology, which is one of the important and well studied aspects in plant
sciences, particularly in the area of signal transduction. Stress is one of the important factors
that significantly influence plant growth index, secondary metabolites production and also
yield.

118 Corresponding Author- Manisha| Copyright, IJSS, 2017

Material and Methods

Seeds of Kenaf (Hibiscus cannabinus) and Hibiscus sabdariffa were collected from local
markets of Andhra Pradesh. Seedlings were raised in pots and then transferred to field for
further growth. Some of the seeds were subjected to surface sterilization.

Chemicals- All the chemicals and solvents used in the present study were purchased from
commercial sources (i.e., SRL chemicals and Rankem chemical Laboratories, Bangalore). All
plant growth regulators used in the present study were procured from Hi-media, Mumbai.

Establishment of in vitro cultures-

For the establishment of in vitro cultures both Kenaf and H. sabdariffa seeds were used.

Seed germination-

Surface sterilization of seeds, surface sterilization is very important procedure before

inoculating seeds for germination to avoid the contamination. In this process, we used
mercuric chloride which is known as bactericide helps to inhibit the growth of bacteria and
bavistin which acts as fungicide and inhibits the growth of fungus. Sterilization of seeds can
be done by these above two microbicide chemicals and with 70% ethanol. Matured seeds of
the above two species were surface sterilized in 70% ethanol for 30 seconds followed by
0.2% bavistin for 5minutes and 0.1% mercuric chloride (w/v) for 1 minute. In this way, seeds
become free from any pathogenic microbes and are able to transfer to the germination media
for its better plant growth without any hazard of contamination. Then the sterilized seeds
were further rinsed for three to four times in sterile distilled water and kept in MS medium
(Murashige and Skoog, 1962) contained in 200ml jars and bottles with sucrose (30gL-1) and
myo-inositol (0.10g/L) and the medium was adjusted to pH 5.8 with sodium chloride, before
autoclaving at 121C for 20min. for germination, the cultures were incubated at 25C in 16h
photoperiod using cool white fluorescent tubes.

119 Corresponding Author- Manisha| Copyright, IJSS, 2017

 Table-1 Components of MS basal medium with minor modifications (Murashige and
Skoog, 1962)

Stocks Chemical Stock Quantity

(g/L) required/Litre
A (50X) Ammonium nitrate 82.50 20.0 ml
B (50X) Potassium nitrate 95.00 20.0 ml
C (200X) Boric acid 1.24 5.0 ml
Potassium dihydrogen phosphate 34.00
Potassium iodide 0.166
Sodium molybdate dehydrate 0.050
Cobalt chloride hexahydrate 0.005
D (200X) Calcium chloride dihydrate 66.40 5.0 ml
E (200X) Magnesium sulfate heptahydrate 74.0 5.0 ml
Manganese sulfate monohydrate 3.38
Zinc sulphate heptahydrate 1.725
Copper sulphate pentahydrate 0.005
F (200X) EDTA disodium salt dehydrate 7.46 5.0 ml
Ferrous sulfate heptahydrate 5.56
G (200X) Glycine 2.0 5.0 ml
Nicotinic acid 0.5
Pyridoxine-HCl 0.5
Thiamine-HCl 0.1
Sucrose 30.0g
myo-inositol 0.10g

Callus induction-

Seedlings emerged after 2 weeks were used to prepare explants for raising in vitro cultures.
The shoot tip explants (1-2 cm), the cotyledonary leaf explants (~1sq cm) and hypocotyl
explants were used in this study. Similarly the one month old leaves from ex vitro plants were
also used for callogenesis after surface sterilization. For initiation of callus, explants were
cultured on MS medium containing various combinations of plant growth regulators such as
2, 4-D (1.0, 2.0, 3.0 mg/L) and kinetin (0.1, 0.5, 1.0 mg/L), combination of 2, 4-D and
kinetin, IAA and kinetin, NAA, BAP and combination of NAA and BAP with 3% sucrose
and 0.8% agar as solidifying agent and the media were adjusted to a pH of 5.8 before
autoclaving. All the cultures were maintained at 16-h photoperiod at 25C (3000 lux)
followed by a dark period of 8 hr.

Explants cultured in HCM media produced a good response with proliferating greenish
friable callus. So HCM media combinations were used for further establishment of cell
suspension cultures.

Preparation of cell suspension cultures-

After three to four subcultures in HCM (NAA, 1mg/L and BA, 4mg/L of M.S media), the
cotyledonary leaf derived callus were sub cultured and transferred to 150ml conical flasks
containing 40ml of liquid HCM suspension medium and the media pH were adjusted to 5.8

120 Corresponding Author- Manisha| Copyright, IJSS, 2017

before autoclaving. Cultures were grown on a rotary shaker at 100rpm with 12 h
photoperiods at 25C.

Initiation of normal root cultures in vitro

The in vitro root cultures were established by sub-culturing the roots derived from callus of
petiole explants. Approximately, 150-200mg of roots were chopped from the explants and
transferred and cultured in 150ml of conical flasks containing 40ml of HCD medium
(MS+NAA 2.0mg/L). The media pH was adjusted to 5.8 before autoclaving and the cultures
were grown on a rotary shaker at 100rpm with 12h photoperiods at 25C.

Table-2 various combination of plant growth regulators used for initiation of callus
cultures.

Label MS+ Plant growth regulators(mg/L)

NAA BAP
HCA 0.1 0
HCB 0.5 0
HCC 1.0 0
HCD 2.0 0
HCE 0.1 1.0
HCF 0.1 3.0
HCG 0.1 4.0
HCH 0.5 1.0
HCI 0.5 3.0
HCJ 0.5 4.0
HCK 1.0 1.0
HCL 1.0 3.0
HCM 1.0 4.0
HCN 2.0 2.0

Analysis of phytoconstituents in leaves of Kenaf

Determination of phenolic content-

500mg of sample was weighed and extracted with 10ml of ethanol (80%). The residue was
centrifuged at 10,000rpm for 20min and extracted with 5ml ethanol was diluted with 5ml of
distilled water. 0.1 ml of extract was pipetted out into a test tube and used for further analysis.
0.1 to 1ml of working standard (catechol 0.1mg/ml) was taken for preparing standard curve.
The volume in all the tubes was made upto 3ml with distilled water. 0.5ml of folin ciocalteau
reagent was added into each test tube and incubated for 3min. 2ml of 20% Na2CO3 solution
was added to each tube. The tube were vortexed and placed in boiling water bath for exactly
1min. the contents were cooled and the absorbance was measured at 650nm and the amount
of phenolics present in the samples was obtained by plotting against the standard graph.

121 Corresponding Author- Manisha| Copyright, IJSS, 2017

Determination of flavonoid content-

By AlCl3 method based on the formation of a complex flavonoid aluminium [1]. The extracts
were diluted with absolute ethanol to an appropriate conc. Then 1ml of diluted sample was
mixed with 1ml of 2% (w/v) methanolic solution of AlCl3. After incubation at room
temperature for 15min., the absorbance of the reaction mixture was read at 430 nm with
spectrophotometer. Rutin was used as standard.

Extraction of ascorbic acid-

Ascorbic acid was extracted (under subdued light) according to the method [2]. with some
modification, from fresh leaves of hibiscus. 1gm of samples was homogenised with 2ml of
methanol and 10ml of cold extraction solution, containing 3% MPA (w/v), 0.05% EDTA
(w/v) and 0.8% glacial acetic acid (v/v). After homogenization, mixture was centrifuged for
15minutes at 5000 rpm at 4C. Then later, supernatant was collected and filtered into HPLC
vials.

Titrimatric quantification method-

Ascorbic acid is oxidised by the blue coloured dye, 2, 6- dichlorophenolindo phenol to

dehydro ascorbic acid at the same time, the dye is reduced to a colourless compound so that
the end point of the reaction easily determined. 2,6- dichlorophenolindophenol (3, 2 mg of
dye was dissolved in about 40ml of hot water, this is cooled and made upto 50ml with
distilled water. 20mg/100 ml solution standard prepared from 5ml of sample and 1ml of
G.A.A (glacial acetic acid). Followed by titration with dye solution, until the blue dye
changes to a permanent pink colour which was visible in 30 seconds.

Chlorophylls and total carotenoids-

Chlorophylls and total carotenoids were measured and characterized by UV-VIS

spectroscopy according to[3]. In an acetone extract, chlorophyll a (chl a) showed the
maximum absorbance at 663nm, chlorophyll b (chl b) at 645nm and total carotenoids
at470nm and the concentrations of chl a, chl b and the sum of carotenoids (cx+c) was
calculated for acetone extraction, where the pigment concentrations are given in g/ml extract
solution.

Chl a (g/ml) = 11.24 A661.6 - 2.04 A644.8

Chl b (g/ml) = 20.13 A644.8 4.19 A661.6

Cx+c (g/ml) = (1000 A470 -1.90 Chl a 63.14 Chl b)/214

Estimation of total antioxidant assay by phosphomolybdenum method-

The total antioxidant activity of extract was evaluated by the phosphomolybdenum assay
method. [4]which is based on a reaction of Mo (V1) to Mo (V) by the extract and subsequent
formation of the green phosphate- Mo (V) complex in acidic condition.

122 Corresponding Author- Manisha| Copyright, IJSS, 2017

A 0.3ml extract (2mg/ml) was combined with 28mm sodium phosphate, 4mm ammonium
molybdate and the reaction mixture was incubate at 95C for 90 minutes then the absorbance
of the solution was measured at 695nm using a visible spectrophotometer against blank after
cooling to RT. The antioxidant activity was expressed as the number of gram equivalent of
ascorbic acid.

Preparation of biotic Elicitors-

The biotic elicitor used in the present study, Rhizopus oligosporus and Aspergillus niger
(obtained from food microbiology department, CFTRI) were maintained on potato dextrose
agar slants. For the preparation of biotic elicitors, the fungal cultures were grown in 2L
conical flasks containing 500ml of potato dextrose broth for 15 days at room temperature
(25C) under dark condition. The fully grown mycelia with spores containing flaska were
autoclaved for 20min a 121C. After autoclaving, the mycelial extract was collected by
filtration and homogenized in a pestle and mortar using acid washed neutralized sand. The
homogenized suspension was centrifuged at 8000g, and after autoclaving for 20minutes at
121C, the supernatant were used as elicitors.

Stock solutions were prepared at a concentration of 0.5, 1 and 2% (w/v) and they were
transferred to the respective root derived cell suspension liquid medium. The cell-suspension
cultures without the inclusion of biotic elicitors were considered as controls. The suspension
cells were harvested at seven days after elicitor application. The fresh and dry weights were
recorded by separating the cells from liquid culture medium by filtration. Each experiment
was carried out with of three replicates.

Influence of biotic elicitors on selective bioactive in in vitro cultures of kenaf-

After filtration, the fresh weight of the root cell suspension cultures was determined and the
extract was used for the analysis of following selective bioactive and methods used were
same as explained above.

Results and discussion-

Screening of in vitro established cultures and in vitro leaves for carotenoid profile was
performed. The total carotenoid content was 0.026% Leaf (ex vitro); 0.015% in in vitro leaf,
0.012% in callus respectively. The total content of leaves was 120-150g/100g F.wt. as
confirmed by HPLC.

Table-3 major phytoconstituents in leaves of Hibiscus sabdarifa

123 Corresponding Author- Manisha| Copyright, IJSS, 2017

 Total carotenoids 0.026% Leaf (ex vitro);

(fresh weight) 0.015% in in vitro leaf, 0.012% in callus,

Total xanthophylls (fresh weight) 0.040% Leaf (ex vitro)

Fresh leaves Total phenolic, content (0.31%);

Total flavonoids (0.391%)

Ascorbic acid content (0.10%)

Total chlorophyll content (0.197%)

Total antioxidant compounds (3.92%)

Dry leaf powder (room dry at 28C) Total phenolic, content (2.5%)

Total flavonoids (0.359%)

Ascorbic acid content (0.45%) Total

chlorophyll content (0.363%) Total
antioxidant compounds (12.11%)

The values are an average of three replicates

There total phenolic content and total antioxidant values are very important for deciphering
functional attributes of leaf of kenaf. These values are on par with the other leafy vegetables
and the contents are significant from health point of view. Similarly flavonoids too contribute
for nutraceutical potential of kenaf leaf. In the present study, the total flavonoids content was
upto 0.39% in leaves. The data presented in the table indicates the changes for different
parameters pertaining to fresh and dry weight basis are agreeable with proportionate increases
of value as the weight in denominator obviously gives more value. But the interesting thing to
observe is that, there was not much loss in respective parameter (phenolics, flavonoids etc),
under room drying conditions.

Initiation of callus cultures of kenaf-

Out of the various combinations of auxins and cytokinins used for initiation of callusing from
leaf explant of H. cannabinus, only the combination of NAA and BAP has shown good
response, hence the same only presented in table 2. On medium containing NAA alone at 0.1
to 1.0mg/L proliferating callusing was evident with sporadic root formation. The callus was
white to light green and hard. At higher levels of NAA (2.0mg/L) profuse rooting was
common in almost all explants. The roots were white, long with root hair too. The root length
was in the range of 0.5 to 4cm in explants that cultured for one month at 16:8 hrs
photoperiod. In complete dark the response for both callusing and rooting was poor. In
combination of NAA and BAP, some sort of response was evident, and the nature of callus
depends on the concentration of respective growth regulator. According 0.5mg/L NAA and
4mg/L BAP showed greenish friable callus. Similarly 1mg/L NAA and 4mg/L produced
friable callus. This indicates the necessity of higher cytokinin and low auxin requirement to
124 Corresponding Author- Manisha| Copyright, IJSS, 2017
get friable callus. The callus that obtained was further sub-cultured onto the same medium
and allowed to grow for one month. After two subcultures the obtained callus was inoculated
into HCA medium broth to get suspension cultures. Accordingly the obtained culture was
sub-cultured twice with 3 weeks interval and used for analysis of phytoconstituents.

Callusing from hypocotyl explants in vitro-

Callusing from in vitro hypocotyl explants is efficient in presence of 2, 4- D (2mg/L) and

kinetin 0.5mg/L. similarly IAA 2.0mg/L and kinetin 0.5mg/L induced green callus from
proximate ends of hypocotyl explants.

Table-4 Callogenesis form leaf explants of H. canabinus

Medium used Plant growth regulator (mg/L) Observation

NAA BAP
HCA 0.1 0 Proliferating callus
rooting very little
HCB 0.5 0
HCC 1.0 0
HCD 2.0 0 Rooting and mild callus
HCE 0.1 1.0 Light green callus
HCF 0.1 3.0 Hard greenish callus
HCG 0.1 4.0 Callusing good, but hard
HCH 0.5 1.0 Light green, puffy callus
HCI 0.5 3.0 Light green callus, friable
HCJ 0.5 4.0 Friable good callus
HCK 1.0 1.0 Friable greenish yellow
callus
HCL 1.0 3.0 Greenish friable very
good proliferating callus
HCM 1.0 4.0 Greenish friable very
good proliferating callus
HCN 2.0 2.0 Good callus

Initiation of in vitro root cultures-

The root cultures that obtained from leaf explants on medium containing NAA 2.0mg/L were
separated from the explants after 1month. These root length was in the range of 0.5cm-5cm.
while transferring them in to same medium without agar (broth) the root were chopped into
~0.5cm in length and allowed to grow 16:8hrs light at 25C and 100rpm for 10 days. A tuft of
roots were evident after one week and the root biomass was good by 10days and lasts upto
30days. But for phytoconstituents analysis 10day old root cultures were harvested. The
reason for this is that, the metabolite profile in general will be very high during the log phase
of growth.

Influence of biotic elicitors on major phytoconstituents of roots of kenaf-

125 Corresponding Author- Manisha| Copyright, IJSS, 2017
Two biotic elicitors i.e. Aspergillus niger and Rhizopus oligosporus mycelial extracts were
used as elicitors for enrichment of ascorbic acid, flavonoids and phenolics content.

After 10days of treatment, respective root cultures upon harvesting showed significant
variation in root biomass and also in bioactive profile compared to control.

The harvested mycelial mass weight was recorded and then mycelial extract prepared,
sterilized and used for the experiments as elicitors. Analysis of TPC, ascorbic acid content,
total flavonoids content in vitro raised root cultures showed moderate to significant
improvement compared to control. Apart from this the concentration of respective elicitor
influenced both the biomass and bioactive compound content. There was drastic reduction
root biomass in all treatments compared to control. Apart from this the concentration of
respective elicitor influenced both the biomass and bioactive compound content. There was
drastic reduction root biomass in all treatments compared to controls. This could be attributed
to the impact of elicitor stress on growth of tissues. But respective bioactives profile
increased at the same time due to elicitor stress. At higher concentration of A. niger (2.0%), it
was almost doubled to that of lower concentration and ten times augmention compared to
control for phenolics and ascorbic acid respectively. A similar observation was also noticed
when Rhizopus oligosporus used as an elicitor. In contrast to phenolics and ascorbic acid
content, there was no increase in flavonoids content. Even at higher dose of elicitor treatment
the content has been reduced compared to control. There was no notable variation or
reduction in TPC and ascorbic acid content in analysed root cultures when they are fresh and
dry.

Table-5 Influence of elicitor treatment on various bioactives production in in vitro

normal root cultures of kenaf

Treatment/Elicitor Root TPC % dry Total Ascorbic acid

biomass (dry wt. flavonoids (% % dry wt.
wt. in mg) F.wt/dry wt.)
A.niger 0.5% 29.3 0.479 0.024/0.185 1.12
A.niger 1.0% 20.7 0.505 0.038/0.258 2.48
A.niger 2.0% 34.4 1.113 0.022/0.328 3.73
R.oligosporus 0.5% 19.6 0.083 0.011/0.118 0.62
R.oligosporus 1.0% 25.0 0.162 0.007/0.055 0.89
R.oligosporus 2.0% 35.0 1.005 0.018/0.206 1.70
Control 50.0 0.059 0.0038/0.035 0.315
Values are an average of triplicates

Elicitor mediated augmentation of secondary metabolites in plants in plants is well

documented particularly in in vitro models of i.e. suspension cultures and root cultures [5][6].
The significant thing in our study is the augmentation of annatto pigment under ex vitro
conditions through floral spray of respective biotic elicitors which is a novel method with
reference to natural colors of any category.

One way of possible mechanism is that the elicitors may act as hormones regulating
secondary metabolism in those plants which are able to decode and further modulate the

126 Corresponding Author- Manisha| Copyright, IJSS, 2017

molecular signal [7]. Our study is in accordance with a similar elicitor spray approaches in
soybean for isoflavones improvement either on fully grown plant [8] or at seeding stage [9].

Production of selective bioactives from normal root cultures that have been reported in this
study is unique from the other root culture reports that produce secondary metabolites like
Hemidesmus indicus [10], Decalepis hamiltonii [11], Angelica gigas [12] antioxidant activity as
in case of Ocimum sanctum [13]. Similarly liposoluble pigment from edible roots of Beta
vulgaris [14] and localization of carotenoids in roots of graminaceous plants [15] was known.
Main difference for the uniqueness is that all the earlier secondary metabolites that are
produced under in vitro normal root cultures were produced in root only in the natural
environment; whereas, in case of kenaf, respective bioactives in our study that are familiar
with leaves in the natural environment were produced under in vitro conditions through
normal root cultures.

Conclusion-

In view of the obtained results, it could be possible to use in vitro raised normal root cultures
as a model for studying the respective pathways of bioactives either by molecular biological
methods or biotransformation. The stress in the form of biotic elicitor that under used under
in vitro conditions in the present study showed significant enhancement of TPC and ascorbic
acid content but there was no surge in case of total flavonoids content. As plants under field
conditions are unprotected to various biotic and abiotic stress factors definitely there will a
noteworthy changes in bioactives contour leaves of kenaf. Under this context, the data
generated in the present study would be having implications to pursue similar studies under
ex vitro conditions to find out the efficacy of biotic elicitors for nutritional improvement of
kenaf.

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128 Corresponding Author- Manisha| Copyright, IJSS, 2017