THE ROLE OF 2, 4 –D AND NAA IN CALLUS INDUCTION OF ACHYRANTHES ASPERA AND ITS SECONDARY METABOLITE...
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Research Article
Journal of Atoms and Molecules An International Online JournalAn International Online JournalAn International Online JournalAn International Online Journal ISSN ISSN ISSN ISSN –––– 2277 2277 2277 2277 –––– 1247124712471247
THE ROLE OF 2, 4 –D AND NAA IN CALLUS INDUCTION OF ACHYRANTHES ASPERA
AND ITS SECONDARY METABOLITE STUDIES
J.Senthilmanickam1, A.Lakshmi Bhavani1*, K.Venkatramlingam 2, G.Chandra2 1PG and Research Department of Biotechnology, Sengunthar Arts and Science College,
Tiruchengode 637205, Nammakal Dist. Tamilnadu, India. 2 Dept. of Zoology, Govt. Arts and Science College, Salem-636007, Tamilnadu, India
Received on: 28-02-2012 Revised on: 04-05-2012 Accepted on: 14–05–2012
Abstract:
In the present investigation callogenic and secondary metabolite studies of Achyranthes aspera
were carried out, stems were used as explants, callus was induced by using two different plant
growth hormones 2,4-D (Dichlorophenoxyacetic acid) and NAA (1-Naphthalene acetic acid) in
different concentration and in combinations supplemented in the MS Medium ( Murashige and
Skoog). The effect of growth hormones (2, 4-D and NAA) in callus induction was studied at the
second and fourth week of culture. During the second week of growth, media supplemented with
the growth hormones 2,4-D and NAA separately showed best response at a concentration of
2.0mg/L and 4.0mg/L respectively. With a combination of both hormones in the media, best
response was seen at the concentration of 2.0+2.0mg/L of 2, 4-D and NAA. Similar results were
observed in the fourth week of culture. Under the morphological character study of the callus
induced by the hormones, A different observation was made with the color of the calli ranging from
large brown to brownish yellow. The presence of steroids, alkaloids and sugars in the callus was
also analyzed by performing thin layer chromatography using various spray reagents accordingly
Key Words: Achyranthes aspera, 2, 4-D, NAA, callus, alkaloids, steroids, sugars, TLC.
Introduction:
Plants have been used in traditional medicine
for several thousand years. The use of
traditional medicine in most developing
countries is a normative basis for the
* Corresponding author
Lakshmi Bhavani A,
Email: [email protected]
Tel: +91 – 9003641450
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maintenance of good health as quoted by
Lucy (Hoareau and Edger Dasila, 1999). The
secondary metabolites of the plants are the
major sources of pharmaceutical, food
additives and fragrances. (Danhanakar et al.,
2000). Medicinal plants have been used as an
exemplary source for centuries as an
alternative remedy for treating human
diseases because they contain numerous
active constituents of immense therapeutic
value (Nostro et al., 2000). In India, herbal
medicines have been the basis of treatment
and cure for various disease and physiological
conditional in traditional methods practiced
such as ayurveda, unani and siddha (Perumal
samy et al., 1998). Asima Chakrabarty and
Adelheid Brantner, (2002) reported the
methanolic extracts of Achyranthes aspera
leaves are valuable antitumor promoter in
cancer. It contains ecdysterone,
dihydroxyketone, alkaloids, saponins and free
tannins. Gokhale et al (2002) showed anti
inflammatory activities of Achyranthes
aspera, in swiss albino mice and inbred wistar
rats. This is due to presence of alkaloid,
saponins and oleanolic acid.
Achyranthes aspera Linn is an abundant
indigenous herb in India and also indigenous
medicinal plant of Asia, South America and
Africa. Commonly used by traditional healer
for the treatment of fever, especially malarial
fever, dysentery, asthma, hypotension and
diabetes. The dried aerial parts are taken
orally in case of diabetes, powder made from
the dried plant is given orally against
whooping cough, decoction of the plant is
used as laxative. Roots of Achyranthes are
used as medicine for diarrhea and cold and the
leaves are used against hypoglycemic activity
and asthma (Rina Chakrabarti and Rao
Vasudeva, 2006). Plant tissue culture method
is used for regeneration and propagation of
plants from unorganized callus tissue derived
from different explants by dedifferentiation
induced by exogenous growth regulators.
Plant regeneration from calli is possible by
denovo organogenesis or somatic
embryogenesis. Callus culture also facilitate
the amplification of limiting plant material.
(Flick and Evans, 1983).The successful
establishment of cell line is capable of
producing high yields of secondary
compounds in cell suspension cultures. The
accumulation of secondary products in plant
cell culture depends on the composition of the
culture medium. In vitro grown plant cells and
tissues have been used extensively for the
production of secondary metabolites. Growth
of the cell in a totally controlled environment
of physical and chemical factors provides an
excellent system for studying changes in the
production of secondary metabolites.
Thin layer chromatography method was used
for qualitative investigation of various groups
of secondary metabolites. Alkaloids are eluted
with different polar solvents like methanol,
chloroform ammonium hydroxide. (Russel
Molyneux and Dale Gardner, 2002). The main
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objective of this study was to initiate callus
from explants of Achyranthes aspera and
check for the presence of secondary
metabolites in the callus extract by TLC.
Materials and methods
Callus induction
Juvenile stems of Achyranthes aspera grown
in the herbal garden of Sengunthar Arts and
Science College, Tiruchengode, were used as
explants source. The explants were washed in
Tween-20 solution for 10min with vigorous
shaking, and then were washed 4 times with
sterile distilled water to remove the excess
Tween-20. The explants were then given a
sodium hypochlorite wash and then washed
with distilled water. Then further disinfection
of the explants were done with 0.1%HgCl2
for 2-5min, rinsed 5 times with sterile
distilled water and then inoculated and
cultured on MS media (Murashige and Skoog,
1962) containing the respective hormones 2,4-
D, (0.5,1.0,2.0,4.0mg/L) NAA
(0.5,1.0,2.0,4.0mg/L) and 2,4-D+NAA
(0.25+0.25, 0.50+0.50,1.0+1.0,2.0+2.0mg/L).
The pH of the medium was adjusted to 5.8
before autoclaving for 20min at 1210C and the
culture tubes were incubated at 25±20C with
55-60% relative humidity and continuous
light.
Analysis of secondary metabolites by TLC
Preparation of TLC Plates:
The adsorbent used for thin layer
chromatography was silica gel G. About 25 g
of silica gel G was taken in a glass mortar and
about 35 ml of the distilled water was added
to it. The mixture was then allowed to swell
for about 15 min. To this an additional 15 ml
of distilled water was added to it with stirring.
The suspension was then spread immediately
on glass plate.
Drying and activation of plates:
The freshly coated plates were then air dried
until the transparence of the layer had
disappeared. The plates were then stacked in a
drying rack and were heated in an oven for 30
min at 110° C. The activated plates were then
used.
Developer:
The developer used were the solvents in the
combination, Toluene: Ethyl acetate: Formic
acid = 3:3:0.5
Preparation of extract using Methanol:
The extracts were prepared using the solvent
methanol. Dry callus of A. aspera were
washed with sterile water and drained, 5 gm
of the dry callus was weighed and
homogenized with 50ml of methanol. The
mixture was transferred to sterile bottles and
kept at room temperature for 24 hours. The
extract was then extracted with a soxlet
apparatus. The mixture was then centrifuged
at 2000 rpm for 10min.The supernatant was
filtered through a sterile funnel containing
sterile whattman filter paper No.1.The filtrate
was stored in sterile bottles at 40C until use.
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Thin Layer Chromatography
Following the spreading of silica gel and the
activation of plates, about 40µ1 of samples
were spotted about 1 cm above the bottom of
the plate and were allowed to dry. The glass
plate was kept in a beaker containing 10ml of
the developer (The samples were not allowed
to immerse in the developer).The plate was
allowed to develop for 5 min till the solvent
front reaches 1 cm below the top of the plate.
The solvent front was marked and allowed to
dry. Visualization reagents were used to stain
the glass plate to observe steroid alkaloids,
steroids, alkaloids and sugars.
Visualization reagents
Meyer reagent: (Mercury (II) chloride-
potassium iodide for steroid alkaloid)
Spray solution I: 13.55 gm mercury II
chloride and 49.8 gm potassium iodide was
dissolved separately each in 20 ml of water.
Both solutions were mixed and made up to
one liter with distilled water. Before spraying
a one part of 17 %Hydrochloric acid was
added to 10parts of this solution.
Spray solution II: 5 gm zinc chloride was
dissolved in 80 ml water; 15ml of 36%
hydrochloric acid was added to it.
Spray solution III: 15% Ammonia solution.
Procedure:
After spraying with solution I, the steroid
alkaloid appears as faint yellow spot. The
chromatogram is rinsed for 10 minutes with
water and, then sprayed with solution II and
subsequently with solution III.
Vanillin sulphuric acid: (For steroid)
Spray reagent: 1gm vanillin is dissolved in
100ml of 97% Sulphuric acid.
Procedure: Following the spraying of the
reagent the chromatogram is heated at 120°C
until the spots attain maximum color
intensity.
Dragendroff reagent: (For alkaloids)
Stock solution: 2.6 gm bismuth carbonate and
7 gms sodium iodide was boiled with 25 ml
glacial acetic acid for a few minutes, and after
12 hours the precipitated sodium acetate is
filtered, then 20 ml of the red brown filtrate is
mixed with 80 ml ethyl acetate and 0.5 ml
water is added and then stored in a dark
bottle.
Spray solution: 10 ml stock solution was
mixed with 100 ml glacial acetic acid and 240
ml ethyl acetate.
Anisaldehyde – Sulphuric acid for Sugars,
Steroid, Terpenes.
Spray solutions: This solution is prepared
freshly before use; 0.5ml solution of
Anisaldehyde is added to 50ml glacial acetic
acid and then 1ml of 97%sulphuric acid is
added to it. Following the spray the plates are
heated to 100-105° C until maximum
visualization of spot. The background may be
brightened by the water vapours. The plant
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constituents, Phenols, Terpenes, Sugar and
Steroid turn Violet, Blue, Red, Grey or Green.
Modified spray solution: For visualization of
sugars, 0.5 ml Anisaldehyde, 9 ml ethanol,
0.5 ml of 95% Sulphuric acid and 0.1ml acetic
acid is mixed freshly before use.
Toluene: Ethyl acetate: formic acid (3:3:0.5
v/v/v) has been identified as the best solvent
system since maximum separation of the
chemical constituent was seen.
Results and Discussion
Callus induction
In the present investigation, two observations
were made to obtain a fine callus initiation
response from stem explants of Achyranthes
aspera. Callus induction by different
hormones supplemented in the MS medium
was studied The effect of the two growth
hormones, 2, 4-D and NAA supplemented in
the media individually as well as in
combination at different concentrations on
callus induction from stem explants of
Achyranthes aspera was carried out. The
results showed that callus response increases
gradually with increasing concentration of the
growth hormones. Similar pattern of work
was carried out by Zenk (1978). The effect of
2, 4-D in combination with BAP
(Benzylaminopurine), IAA (Indole Acetic
acid), IBA (4- Indol- 3-butyric acid) using
leaf explants of Achyranthes aspera was
studied by Sarakayani Mohammed Zia and
Sadia Sarwar (2008). The increasing use of
plant extracts in the food, cosmetic and
pharmaceutical industries suggests that in
order to derive active compounds, systematic
study of medicinal plant is very important
(Ncube et al., 2008)
Minimum number of callus was observed in
the two week old culture containing 2, 4-D
and NAA separately in the concentration of
0.5,1.0,2.0,4.0mg/L. A low percentage of
callusing was recorded and callus response
was also less significant in the medium with
less concentration of hormones. At the same
time, increased callus response was observed
in the tube containing high concentration of 2,
4-D and NAA (2.0+2.0mg/L) separately and
also in tubes with media containing NAA
individually at a concentration of 4.0mg/L
(Table-1).
Similarly in the four weeks old culture, a
better callus response was observed in all
culture vessels. Percentage of callusing
increased gradually with increasing
concentration of 2,4-D. Maximum 80% of
callus response was recorded at 2.0 and
4.0mg/L of 2,4-D and minimum of 40%
callusing appeared at concentration of
0.50mg/L of 2,4-D. a light decreasing level of
callus response was observed in NAA
containing tubes. Maximum response of 60%
was recorded in culture vessel containing
NAA and minimum of 20% response was
observed at low concentration of NAA. A
high significant response was obtained in the
callus response at low concentration of 2, 4-D
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+ NAA (0.25+0.25, 0.50+0.50mg/L) and
100% response at high concentration of 2, 4-
D + NAA (1.0+1.0, 2.0+2.0mg/L) was
observed (Table-2).
In the observation for morphological
character of callus with the two week and
four week old culture small brownish color
callus was noticed at a concentration of 2,4-D,
(0.50mg/L). Similarly, moderate, brownish
yellow, large and brown color callus appeared
in the concentration of 1.0, 2.0mg/L and
4.0mg/L of 2,4-D respectively.
A different morphological character was
observed in NAA containing culture vessel.
Moderate sized, brownish white, brownish
yellow and brownish green color callus was
observed in the concentration of 0.50, 1.0,
2.0mg/L of NAA respectively. At high
concentration of NAA (4.0mg/L), large and
yellowish green color callus had appeared.
Similar result of callus induction by NAA in
other genera was reported by Mischenko and
Fedoreyev (1999). But in combination of 2,4-
D with NAA, large greenish brown, yellowish
green color callus appeared at different
concentration (0.25+0.25, 0.50+0.50, 1.0+1.0,
2.0+2.0mg/L), (Table-3).
Thin layer chromatography
Thin-layer chromatography continues to be an
important method for qualitative analysis of
steroids because of its inherent advantages-
many samples can be analyzed
simultaneously and quickly, and multiple
separation techniques and detection
procedures can be applied. (Bhawani et al.,
2010). Achyranthes aspera contains
alkaloids, flavanoids, saponins, steroids and
terpenoids (Niranjan Sutar et al., 2011). Out
of which the water soluble alkaloids possess
anti-inflammatory activity. For the analysis of
secondary metabolite, fresh dried callus were
subjected to extraction in a solvent apparatus
with methanol and this sample was used for
chromatography studies. The present study
investigates the presence of secondary
metabolites in the callus from stem explants
of Achyranthes aspera by thin layer
chromatography. The presence or absence of
various secondary metabolites are confirmed
using different visualization reagents viz
Mayer’s reagent, Vanillin sulphuric acid
solution, Dragendroff reagent, Anisaldehyde
sulphuric acid solution (Fig 1, 2, 3&4).
Similar work was studied by various workers
Russell Molyneux and Dale Gardner (2002)
and Peter Houghton (2002). The current
studies reported that steroid alkaloids and
sugars were found in the callus of
Achyranthes aspera and other compounds like
phenols and terpenes were not found in the
callus.
Russell Molyneux and Dale Gardner (2002)
used thin layer chromatography method for
qualitative investigation of various groups of
secondary metabolites. Alkaloids are eluted
with different polar solvents like methanol,
chloroform and ammonium hydroxide. Thus
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appropriate level of 2,4-D and NAA are able
to induce the formation of calli from stem
explants of Achyranthes aspera and this callus
effectively produces alkaloids of steroid type
and sugars which has been revealed by the
work carried out.. The result may further be
exploited for production of certain secondary
metabolites by callus culture and may also be
useful for other tissue culture technique. In
future, a modern analytical method like
HPLC, HPTLC can be employed to identify
the presence of other secondary metabolites
and specific type of alkaloid in the callus of
Achyranthes aspera.
Acknowledgements
The authors are obliged to the management of
Sengunthar Arts & Science College,
Tiruchengode for providing the facilities
without restriction in the laboratory.
S. no. Hormones Concentration
Mg/L
No. of
culture
vessels
Culture
vessel with
callus
% of
callusing
1. 2,4-D
0.5
1.0
2.0
4.0
5
5
5
5
1
1
2
2
20
20
40
40
2. NAA
0.5
1.0
2.0
4.0
5
5
5
5
1
1
2
3
20
20
40
60
3. 2,4-D+NAA
0.25+0.25
0.50+0.50
1.0+1.0
2.0+2.0
5
5
5
5
1
2
2
3
20
40
40
60
Table-1 Effect of plant growth hormones 2, 4-D and NAA on second week old culture of
Achyranthes aspera.
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S. no. Hormones Concentration
Mg/L
No. of
culture
vessels
Culture
vessel
with
callus
% of
callusing
1. 2,4-D
0.5
1.0
2.0
4.0
5
5
5
5
2
3
4
4
40
60
80
80
2. NAA
0.5
1.0
2.0
4.0
5
5
5
5
1
1
3
3
20
20
60
60
3. 2,4-D+NAA
0.25+0.25
0.50+0.50
1.0+1.0
2.0+2.0
5
5
5
5
3
3
5
5
60
60
100
100
Table-2 Effect of plant growth hormones 2, 4-D and NAA on fourth week old culture of
Achyranthes aspera.
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S. no. Hormones Concentration
Mg/L Callus size
Morphological
character
1. 2,4-D
0.5
1.0
2.0
4.0
Small
Medium
Medium
Large
Brownish white
Brownish yellow
Brownish yellow
Brown
2. NAA
0.5
1.0
2.0
4.0
Medium
Medium
Medium
Large
Brownish white
Brownish yellow
Brownish green
Yellowish green
3. 2,4-D+NAA
0.25+0.25
0.50+0.50
1.0+1.0
2.0+2.0
Large
Large
Large
Large
Yellowish green
Greenish brown
Yellowish green
Yellowish green
Table-3 Callogenic response of Achyranthes aspera at different concentration and combination
of 2, 4-D and NAA.
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Presence of
Alkaloids
(Yellow colour)
TLC Plate Figure 1 : Identification of alkaloids by Mayer’s Reagent
Presence of
Steroid
alkaloids (Dark
Yellow colour)
TLC Plate Figure 2: Identification of Steroid type alkaloids by Vanillin sulphuric acid spray solution
Presence of
Alkaloid
(Orange colour)
TLC Plate Figure 3 : Identification of alkaloid by Dragendroff reagent Presence of Steroid (Green Colour) Presence of Sugar (Red colour) TLC Plate Figure 4: Identification of Steroids and Sugars by Anisaldehyde – Sulphuric acid spray solution
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