Pigment Analysis and Tissue Culture of Amaranthus cruentus L.
J.S. Yaacob, L.C. Hwei and R.M. Taha
Institute of Biological Sciences
Faculty of Science
University of Malaya
50603 Kuala Lumpur
Malaysia
N.A. Mat Nor and N. Aziz
Department of Physics
Faculty of Science
University of Malaya
50603 Kuala Lumpur
Malaysia
Keywords:
Abstract
Amaranthus cruentus or the ‘red amaranth’ or the ‘Mexican grain amaranth’
is a flowering plant that belongs to the Amaranthaceae family. It produces edible
seeds that are eaten as a cereal grain or cooked into a porridge, and the leaves are
cooked like spinach. In this study, tissue culture of Amaranthus cruentus was
conducted through manipulation of growth regulators and plant hormones to
produce coloured callus. Hormones such as Naphtalene acetic acid (NAA), Benzyl
aminopurine (BAP), 2,4-Dichlorophenoxyacetic Acid (2,4-D), Indole-3-acetic acid
(IAA), Kinetin, Zeatin and Gibberellic acid (GA3) with different concentrations and
combinations were used. Leaves and stems of Amaranthus cruentus were cultured on
Murashige & Skoog (MS) media, supplemented with different hormone
combinations and incubated under 24-26C with 16 hours of light and 8 hours of
dark to produce cream, red and green coloured callus. The most optimum media for
the induction of red callus was MS media supplemented with 1.5 mg/L IAA and 1.5
mg/L Zeatin, while the most responsive explant was the leaves. The effects of pH and
sucrose content on callus formations were also studied. The results obtained showed
that pH 5.8 and sucrose content of more than 30g/L produced the most optimum
outcome. The red pigments were extracted and analyzed through thin layer
chromatography, simple colour testing and UV/Vis spectrophotometer. The tests
confirmed that the red pigments were flavonoids called betacyanins.
INTRODUCTION
Amaranthus cruentus or the ‘red amaranth’ or the ‘Mexican grain amaranth’ is a
flowering plant that belongs to the Amaranthaceae family. It is an annual, pseudo-cereal
broadleafed plant that is used as a high-protein grain, leafy vegetable, or forage crop. It
produces edible seeds that are eaten as a cereal grain or cooked into a porridge, and the
leaves are cooked like spinach. Amaranth is drought tolerant and requires warm growth
conditions for germination (suitably 65°-75° F).
Coloured Amaranth contains “amaranthine”, which is a constituent of a larger
group called betacyanins (Mabry and Dreiding, 1968). The Amaranth betacyanin had
been identified as 5-0-[-2-0-(b-D– glycopyranosyluronic acid) b-D- glucopyranoside],
which is a betanidine (Piatelli et al. 1964). Natural pigments such as betacyanins and
anthocyanins have been commercially used as food colourants (Von Elbe et al., 1983).
For example, betacyanins from beetroots (Beta vulgaris) has been a major source of
betacyanins, however other plant sources have been widely researched to serve as an
alternative (Piatelli et al., 1965; Lee et al., 1986; Lehmann, 1990; Cai et al., 2001). The
present study reports on efficient generation of coloured callus of Amaranthus cruentus
via tissue culture methods and identification of the coloured pigments extracted from
Amaranthus cruentus callus.
MATERIALS AND METHODS
Production of Coloured Callus of Amaranthus cruentus
The seeds of Amaranthus cruentus were obtained from Singapore and used for invitro cultures, where the seeds were cultured on Murashige and Skoog (MS) media after
surface sterilization with chlorox. The generated leaf, stem and root explants were then
cultured on MS media (plus 30 g/L sucrose and 8 g agar, at pH 5.8) supplemented with
different concentrations of NAA, BAP, IAA, Zeatin, Kinetin, 2,4-D and GA3 to initiate
callus production. The cultures were incubated under 24-26 C with 16 hours of light and
8 hours of dark.
Thin Layer Chromatography (TLC) Test
The red pigments from the red coloured callus (20 g) were solvent extracted using
distilled water at 70 C and centrifuged at 5000 rpm for 15 minutes. The red extract (the
supernatant) was subjected to thin layer chromatography test for 15 minutes in a closed
chamber. A solvent system consisting of butanol, acetic acid and water (BAW) at a ratio
of 4:1:5 was used. After the solvent front had moved 15 cm, the plate was dried at
ambient temperature and protected from light. The resulting coloured spots were marked.
The thin layer chromatography plate was also observed under UV light at a wavelength of
254 nm and 366 nm, and the observed spots were also marked. The plate was then tested
with iodine and the resulting brown spots were marked. Other than that, the plate was
stained with anisaldehyde-sulfuric acid at 100 C, which is a flavonoid reagent, and the
resulting red spots that indicate the presence of flavonoids were marked.
Extraction of Flavonoids from Dried Callus for Pigments Identification
The red coloured callus of Amaranthus cruentus were dried at 45 C for 48 hours
and homogenized. The red pigments from the callus were solvent extracted using
methanol, with dry weight to volume ratio of 1:10. The extracts were then subjected to
several tests including pH, ammonia, mineral acid, acidification and alkalization test and
UV-Visible spectroscopic analysis to determine its identity.
Effects of pH on Coloured Extracts
3 ml of the coloured extract was added into a test tube and the pH of the extract
was varied by addition of NaOH and HCl, to observe the effects of pH on the colour of
the extracts.
Reaction with Ammonia
3 ml of the coloured extract was added into a test tube and soaked in cold water,
followed by addition of 1 ml of 1 M ammonium solution. Any colour variations vary
were observed and recorded.
Reaction with Mineral Acid
3 ml of the coloured extract was added into a test tube and soaked in cold water,
followed by addition of 1 ml of 1 M HCl. Any colour transitions were observed. The
extractions were then heated and the results were recorded. A second test was also done,
which is similar to the first test, with an addition of 2 drops of 1 M nitric acid before
observing the colour changes.
Acidification and Alkalinization Test
2 drops of 1 M sodium hydroxide (NaOH) was added into a test tube containing 3
ml of the coloured extract. Any colour change was carefully observed and recorded. This
is followed by an acidification test, where 2 drops of 1 M hydrochloric acid (HCl) were
immediately added into the test tube. Any colour changes were also observed. A second
test was also done, which is similar to the first test, with an addition of 10 minutes
waiting time before acidification was conducted. Any colour changes were also observed
and recorded.
UV-Visible Spectroscopy
The coloured extracts were subjected to a UV-Visible spectroscopic analysis using
a Shimadzu UV-3101 PC Spectrophotometer, between the wavelengths of 200 nm to 800
nm.
RESULTS AND DISCUSSION
Production of Coloured Callus of Amaranthus cruentus
Leaves and stems of Amaranthus cruentus were cultured on Murashige & Skoog
(MS) media supplemented with different hormone combinations and incubated under 2426C with 16 hours of light and 8 hours of dark. Manipulation and combination of
hormones such as NAA, BAP, 2,4-D, IAA, Kinetin, Zeatin and GA3 had successfully
produced coloured callus (cream, green and red) after 2 weeks of culture, except for the
explants cultured on MS media with the addition of only 2,4-D and explants cultured on
MS media with higher 2,4-D to Kinetin ratio.
In terms of the production of coloured callus, it was found out that the
combinations of 2,4-D and Kinetin (Fig. 1a and 1b), and Zeatin and GA3 produced cream
coloured callus for all explants (Fig. 1c and 1d). Similar observations were made for
explants cultured on MS media with 2,4-D and BAP (Fig. 1e and 1f), but the cream callus
changed its colour to green after 3 weeks of culture. On the other hand, combinations of
NAA and BAP produced cream and creamy-pink coloured callus for both leaf and stem
explants (Fig. 2), but only produced cream coloured callus for root explants. As for the
green coloured callus, this was produced when the explants were cultured on MS media
with only IAA (Fig. 3).
However, the pigment of interest in this study is the red-violet pigment; therefore
optimum production of red callus was aimed. Combinations of IAA and Zeatin produced
red callus for leaf explants, and creamy-red for stem explants (Fig. 4), while MSO (MS
media without any additon of hormones) produced red callus for both leaf and stem
explants, and cream coloured callus for root explants. The most optimum media for the
induction of red callus was MS media supplemented with 1.5 mg/L IAA and 1.5 mg/L
Zeatin (Fig. 4a and 4b), where the red callus obtained were larger in size and had higher
fresh weight average (0.48 g).
Other than the production of coloured callus, growth of adventitious roots was
also observed for explants cultured on MS media supplemented with hormones,
especially when the concentrations of auxin used was higher than 1.0 mg/L (for stem and
leaf explants), but more of these occurrences were observed for MS media supplemented
with NAA, than MS media supplemented with IAA (Fig. 5).
The effects of pH and sucrose content on callus formations of Amaranthus
cruentus were also studied. The results obtained showed that pH 5.8 and sucrose content
of more than 30g/L produced the most optimum outcome (data not shown). Other than
that, it was found out that the most responsive explant was the leaves. This is apparent in
the results obtained, where leaf explants showed 40-100% of callus production, while
stem and root explants produced 35-80% and 15-75% of callus production respectively.
These results are depicted in Fig. 6.
Identification of the Red-Violet Pigment of Amaranthus cruentus Callus
The detection of pigment derived from the callus of Amaranthus cruentus by
using TLC method with BAW solvent shows positive results. There were two red spots
appeared on the TLC plate after the test was completed, which indicates the presence of
betalain. In addition, upon observation under a UV lamp, one of the spots exhibits a violet
fluorescence at 254 nm while the other spot appeared under blue fluorescence region (at
366 nm). The fluorescence spots proved the presence of aromatic compounds, responsible
in giving the Amaranthus cruentus callus its distinct red colour. The colour of the extract
is as depicted in Fig. 7.
In the acidification and alkalinization tests, the red-violet colour of the extract
changed to brown and immediately into yellow after addition of 1 M of NaOH, and
changed back to its original red-violet colour after the acidification process. However, in
the second test where 10 minutes of waiting time was applied before acidification, the
sample colour remained yellow and did not turn into its original colour (red-violet), even
after addition of HCl. This is a distinct characteristic of a betalain compound, which is a
class of pigment that constitutes of two groups, namely the betacyanin and betaxanthin
(Tsai et al., 2010). To date, 50 types of betacyanin (red-violet in colour) and 20 types of
betaxanthin (yellow in colour) had been identified (Tsai et al., 2010). This therefore
provides the preliminary evidence that the coloured pigment of Amaranthus cruentus
callus is a betalain compound, specifically a betacyanin.
The tests with ammonia and mineral acid also gave positive results, where the redviolet colour of the extract remains unchanged after addition with ammonia. However, in
the mineral acid tests, the red-violet colour became darker in cold conditions and will
fade in heated conditions upon addition with HCl, but will fade in both cold and heated
conditions upon additon of nitric acid. These additional three observations further confirm
the identity of the coloured pigment as betacyanin.
Other than that, the pH variation tests also reaffirmed the identity of the red-violet
pigment as betacyanin. The red-violet colour of the extract turned yellowish-brown in
alkaline conditions and turned red in acidic conditions, but remained red-violet in colour
in neutral conditions, which again is a distinct characteristic of betalain.
Moreover, betacyanins exhibit a maximum absorbance at wavelengths ranging
from 532 nm to 554 nm (Harborne, 1967). In this study, the UV-Visible spectroscopic
analysis results showed a maximum absorbance of 0.1 at 536 nm (Fig. 8), therefore
confirming the identity of the red-violet pigment of Amaranthus cruentus callus as a
betacyanin.
CONCLUSIONS
The production of callus and their colours are influenced by several factors, such
as the differences in hormone combinations and concentrations used and the explant
source. Combinations of 2,4-D and Kinetin, Zeatin and GA3 and 2,4-D and BAP would
produce cream callus, while combinations of NAA and BAP, IAA and Zeatin and only
IAA would produce creamy-pink callus, red callus and green callus respectively.
However, the most optimum media to produce red callus was MS media supplemented
with 1.5 mg/L IAA and 1.5 mg/L Zeatin.
Both the physical and chemical characteristics tests conducted on the red-violet
extracts of Amaranthus cruentus indicate the presence of flavonoids called betacyanins.
ACKNOWLEDGEMENTS
The authors would like to thank the University of Malaya for financial assistance
and facilities provided.
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Figures
Fig. 1. Production of cream callus after 4 weeks of culture on MS media supplemented
with hormones (a leaf in 0.5 mg/L 2,4-D and 1.5 mg/L Kinetin, b leaf in 1.5 mg/L
2,4-D and 0.5 mg/L Kinetin, c leaf in 1.0 mg/L GA3 and 1.5 mg/L Zeatin, d root in
1.0 mg/L GA3 and 1.5 mg/L Zeatin, e leaf in 3.0 mg/L 2,4-D and 0.5 mg/L BAP,
f stem in 3.0 mg/L 2,4-D and 0.5 mg/L BAP).
Fig. 2. Production of creamy-pink callus after 4 weeks of culture on MS media
supplemented with NAA and BAP (a leaf in 0.5 mg/L NAA and 1.5 mg/L BAP,
b stem in 0.5 mg/L NAA and 2.0 mg/L BAP, c leaf in 1.5 mg/L NAA and 1.0
mg/L BAP, d stem 1.5 mg/L NAA and 1.0 mg/L BAP).
Fig. 3. Production of green callus on leaf explants after 4 weeks of culture on MS media
supplemented with 1.5 mg/L IAA.
Fig. 4. Production of red callus after 4 weeks of culture on MS media supplemented with
IAA and Zeatin (a stem in 1.5 mg/L IAA and 1.5 mg/L Zeatin, b leaf in 1.5 mg/L
IAA and 1.5 mg/L Zeatin, c leaf 1.5 mg/L IAA and 1.0 mg/L Zeatin).
Fig. 5. Generation of red and white adventitious roots on green callus, produced after 4
weeks of culture of stem explants on MS media supplemented with 1.5 mg/L IAA.
Fig. 6. Effects of different hormone combinations on the production of Amaranthus
cruentus callus (%).
Fig. 7. Red-violet extract obtained from solvent extraction of the Amaranthus cruentus
red callus.
Fig. 8. UV-Visible spectrum profile of Amaranthus cruentus betacyanin extracts.