Pigment Analysis and Tissue Culture of Amaranthus cruentus L




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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
N.A. Mat Nor and N. Aziz

Department of Physics

Faculty of Science

University of Malaya



50603 Kuala Lumpur

Malaysia
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 in-vitro 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 24-26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 red-violet 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.


Literature Cited

Cai, Y., Sun, M. and Corke, H. 2001. Identification and distribution of simple and acylated betacyanins in the Amaranthaceae. J. Agric. Food Chem. 49:1971-1978.

Harborne, J.B. 1967.Comparative Biochemistry of the Flavonoids. London: Academic Press.

Lee, S.Y., Shin, Y.C., Byun, S.M., Jo, J.S. and Cho, S.J. 1986. Evaluation of red pigment of cockscomb flower: color stability of the red pigment. Korean J. Food Sci. Technol. 18:388-392.

Lehmann, J.W. 1990. Pigments of grain and feral amaranths. Legacy. 3(1):3-4.

Mabry, T.J. and Dreiding, A.S. 1968. The betalains. Recent Advances in Phytochemistry. 1:145-160.

Piatelli, M., Minale, L. and Prota, G. 1964. Isolation and structure of amaranthine and isoamaranthine. Ann. Chim. 54:963-968.

Piatelli, M., Minale, L. and Prota, G. 1965. Pigments of Centrospermae III. Betaxanthins from Beta vulgaris L. Phytochemistry. 4:121-125.

Tsai, P.J., Sheu C.H., Wu P.H. and Sun Y.F. 2010. Thermal and pH Stability of Betacyanin Pigment of Djulis (Chenopodium formosanum) in Taiwan and Their Relation to Antioxidant Activity. J. Agric. Food Chem. 58(2):1020-1025.

Von Elbe, J.H., Schwartz, S.J. and Attoe, E.L. 1983. Using appropriate methodology to predict food quality. Food Technol. 37:87-91.



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.




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