Callus Induction and Influence of UV-C on Betalain Content in the Callus of an Amaranth

Callus induction and influence of UV-C on betalain content in the callus of an amaranth

Received for publication, September 01, 2014 Accepted, July, 14, 2015

KITTI BODHIPADMA1, SOMPOCH NOICHINDA1, PHETCHARAT WACHIRABONGKOTH1, ANGKANA THONGRUANG1, KORAVISD NATHALANG2, YOSSAPOL PALAPOL3 and DAVID W. M. LEUNG4* 1Division of Agro-Industrial Technology, Faculty of Applied Science, King Mongkut’s University of Technology North Bangkok, Bangsue, Bangkok 10800 Thailand 2 Department of Biology, Faculty of Science, Mahidol University, Rama VI Rd., Rajathevi District, Bangkok 10400, Thailand 3 Division of Agricultural Technology, Faculty of Science and Arts, Burapha University, Chanthaburi Campus, Thamai District, Chanthaburi 22170, Thailand 4 School of Biological Sciences, University of Canterbury, Private Bag 4800, Christchurch 8140, New Zealand *Address for correspondence to:School of Biological Sciences, University of Canterbury, Private Bag 4800, Christchurch 8140, New Zealand;E-mail: [email protected]

Abstract Hypocotyl explants of three amaranths, Amaranthus cruentus L., Celosia argentea var. plumosa (Burvenich) Voss and Gomphrena globosa L., were cultured on agar-gelled Murashige and Skoog (MS) medium supplemented singly with 0, 0.5, 1 and 2 mg/l of 2,4-D, NAA or BAP in the light for 4 weeks. No callus formed from hypocotyl explants of C. argentea var. plumosa and G. globosa on media supplemented with 2,4-D and BAP, respectively. Calli were, however, formed by hypocotyl explants of the three species on NAA-containing media. Only C. argentea var. plumosa hypocotyl explants on BAP- supplemented media formed red compact callus, which exhibited the highest increase (4 fold) in fresh weight after 4 weeks on media containing 0.5 mg/l BAP compared to 1 or 2 mg/l BAP. Both betacyanins and betaxanthins were detected in the red callus and their levels could be elevated when the hypocotyl explant was irradiated with UV-C before callus initiation or the callus was irradiated directly.

Keywords:Amaranthus, BAP, Celosia, Gomphrena, hypocotyls, red pigments

1. Introduction Currently, the demand for natural colouring substances is increasing not only in food industry but also in medicinal, pharmaceutical and cosmetic products [1-3]. Betalains, the water-soluble colourants, are not only thought to play significant roles in plant physiology and reproduction but also in human nutrition as well [4, 5]. In nature, betalains are found in only 10 families of the core Caryophyllales, for example, Amaranthaceae and Cactaceae [6- 8]. Biosynthesis of the two types of betalains, the red-violet betacyanins and the yellow- orange betaxanthins, starts from the amino acid L-tyrosine [9, 10]. These nitrogen-containing pigments have generally been regarded as safe food colourants which do not require any chemical modifications for utility in a wide variety of manufactured goods and do not give any undesirable flavours to foods [1]. The well-known genera of the Amaranthaceae family in Thailand are Amaranthus, Celosia and Gomphrena. Among these plants, Celosia argentea var. plumosa (Burvenich) Voss, C. argenteavar. cristata (L.) Kuntze and Gomphrena globosa L. are attractive plants

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KITTI BODHIPADMA, SOMPOCH NOICHINDA, PHETCHARAT WACHIRABONGKOTH, ANGKANA THONGRUANG, KORAVISD NATHALANG, YOSSAPOL PALAPOL, DAVID W. M. LEUNG that have been mainly used for landscape decoration throughout the country while A. cruentus L. is preferred as ornamental plants and in Thai cuisine. Being in the Amaranthaceae family these plant species are expected to produce betalains. Plant tissue culture technology has been shown to be an alternative approach for the production ofbetalains in some plants under aseptic conditions [11, 12]. It was also found that different types of ultraviolet (UV) radiation enhanced the production of some important secondary compounds. For example, UV-A induced biosynthesis of anthocyanin in Brassica rapa hypocotyls, UV-B stimulated the production of flavonoid in Passifloraquadrangulariscallus and UV-C increased the quantity of stilbenoids in Arachishypogaea callus [13-15]. Here, we investigated the possibility of betalain production in callus cultures of A. cruentus, C. argentea var. plumosa and G.globosa and whether UV-C irradiation could enhance betalain production in these cultures. An initial objective was to examine the effects of selected plant growth regulators (PGRs) on callus induction and growth from the hypocotyl explants of these three plants. In the second part of this investigation, only calli with red pigmentation were subcultured for determination of betalain contents and the effects of treatment with UV-C irradiation.

2. Materials and Methods Plant materials and surface sterilization Seeds of Amaranthus cruentus were purchased from HI-Q Agricultural Co., Ltd. Bangkok, Thailand while Celosia argentea var. plumosa and Gomphrena globosa seeds were bought from Thai Seed and Agriculture Co., Ltd. Bangkok, Thailand. These seeds were first soaked for 3 h in distilled water and then for 15 min in 15% (v/v) Clorox (a commercial bleach solution containing 5.25%, w/w, sodium hypochlorite as available chlorine) to which 2-3 drops of Tween-20 were added. After this, they were rinsed three times with sterile distilled water (3 min for each rinse) and then the surface-sterilized seeds were transferred to basal Murashige and Skoog (MS) medium [16] for seed germination and kept in a growth room under 16 h illumination from daylight fluorescent lamps (26.5 μmol m−2 s−1 light intensity) and 8 h darkness at 25±2 °C for 7 days. All media were adjusted to pH 5.7, dispensed into glass culture vessels (4.5 cm width x 6.5 cm height), gelled with 0.9% (w/v) agar, and autoclaved at 121 °C and 103 kPafor 20 min.

Effects of plant growth regulators (PGRs) on callus induction and proliferation After seed germination under aseptic conditions, hypocotyl explants (about 1 cm from the middle of the hypocotyl closer to the root) from the seedlings were excised and placed horizontally on the surface of culture medium in culture containers (one hypocotyl explant placed in a container) with one of the following media: basal MS medium supplemented with 0, 0.5, 1 and 2 mg/l of a PGR (2,4-D, or NAA or BAP). Cultures were kept in a growth room for 4 weeks under the same conditions as those for seed germination described above. For the experiment investigating the betacyanin and betaxanthin contents in callus extracts, only callus with red pigmentation was excised and separated from the hypocotyl explants before subculturing onto the same callus induction media for 4 weeks.

Effects of UV-C on betacyanin and betaxanthin levels The effects of UV-C irradiation treatments on betacyanin and betaxanthin contents in callus extracts were investigated. In the first treatment, the hypocotyl explants of C. argentea var. plumosa was irradiated with a UV-C radiation source (a Philips TUV 15 watt T8 UV-C

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Callus induction and influence of UV-C on betalain content in the callus of an amaranth germicidal light bulb [G15T8] installed in a laminar flow cabinet emitting radiation at 253.7 nm) prior to callus initiation and proliferation. In the second treatment following the red callus induction from the hypocotyl explants after 4 weeks, the callus was excised from the explants before it was exposed to UV-C radiation. In the respective treatments, the sterile containers, each with one hypocotyl explant or a piece of red callus, were placed inside the laminar flow cabinet before the UV-C light was turned on for 15 min. After this, the irradiated hypocotyl explants were cultured for callus initiation and the UV-C irradiated callus was subcultured. The control was callus which was initiated from non-irradiated hypocotyl explants and was not irradiated during culture. The betacyanin and betaxanthin levels in the extracts of the red calli from the different treatments were quantified as described below.

Determination of betacyanin and betaxanthin levels Betacyanin and betaxanthin contents in red callus were extracted and quantified according to the method of von Elbe [17]. Freshly harvested red callus (1 g; n=4) from one of the UV-irradiation treatments at a given time of subculture was ground and extracted with three ml of distilled water at 4 °C. The homogenate was transferred to a Buchner funnel and filtered through Whatman No. 1 filter paper and the filter cake was washed several times with distilled water until the filtrate was colourless. The volume of the filtrate was adjusted to five ml before absorbance was determined using a spectrophotometer at 476, 538 and 600 nm. The following formulae were used for calculation of: betacyanin content = [(x× DF) / 1120] × 1000 mg/gFW callus betaxanthin content = [(y× DF) / 750] × 1000 mg/gFW callus (x = 1.095 × (a − c), z = a –x, y = b − z − x/3.1) Where a = light absorption of the sample at 538 nm b = light absorption of the sample at 476 nm c = light absorption of the sample at 600 nm x = light absorption of betacyanins y = light absorption of betaxanthin z = light absorption of impurities (DF is the dilution factor.)

Statistical analysis For all the experiments, there were six replicate culture jars each containing one hypocotyl explant or one piece of callus. ANOVA (at P < 0.05) of data and the differences in means were compared using DMRT (Duncan’s New Multiple Range Test)at P < 0.05. The computer software program used for all these data analyses was SPSS version 11.5.

Table 1. Effect of different concentrations of BAP on callus growth of Celosia argenteavar. plumosaunder light during 4 weeks of subculture. Time (weeks) Fresh weight (mg per hypocotyl explant( MS + 0.5 mg/l BAP MS + 1 mg/l BAP MS + 2 mg/l BAP 0 325.5 ± 2.1a 312.2 ± 2.5a 309.8 ± 2.2a 1 663.3 ± 11.2a 557.8 ± 34.5b 554.7 ± 7.8b 2 743.8 ± 20.5a 678.7 ± 4.8b 638.0 ± 20.4b 3 925.9 ± 18.1a 836.7 ± 21.8b 788.5 ± 26.0b 4 1161.9 ± 14.7a 1051.3 ± 13.8b 901.8 ± 27.4c Values are mean fresh weights of calli per 6 replications (6 jars each containing one hypocotyl explant) ± SE. Data marked by same letter in each row are not significantly different (ANOVA, P< 0.05). Romanian Biotechnological Letters, Vol. 22, No. 4, 2017 12795

KITTI BODHIPADMA, SOMPOCH NOICHINDA, PHETCHARAT WACHIRABONGKOTH, ANGKANA THONGRUANG, KORAVISD NATHALANG, YOSSAPOL PALAPOL, DAVID W. M. LEUNG

Table 2. Changes in betacyanin contents in calli of Celosia argenteavar. plumosa during 4 weeks of subculture. Control: callus was initiated and then subcultured on MS medium supplemented with 0.5 mg/l BAP under light); Hypocotyl-UV: hypocotyl explants were irradiated with UV-C before callus induction and proliferation; Callus- UV: callus that was initiated from non-irradiated hypocotyl explants and was irradiated with UV-C before subculture. Time (weeks) Betacyanin content (mg/g FW) control Hypocotyl-UV Callus-UV 0 0.36 ± 0.03b 0.57 ± 0.01a 0.36 ± 0.03b 1 0.36 ± 0.01c 0.77 ± 0.03a 0.46 ± 0.03b 2 0.37 ± 0.05c 1.04 ± 0.14a 0.76 ± 0.08b 3 0.61 ± 0.05b 1.18 ± 0.10a 1.05± 0.16a 4 0.70 ± 0.04b 1.39 ± 0.03a 1.25 ± 0.07a Values are mean ± SE from 4 replications of analysis. Data marked by same letter in each row are not significantly different (ANOVA, P< 0.05).

Table 3. Changes in betaxanthin contents in calli of Celosia argenteavar. plumosa during 4 weeks of subculture. Control: callus was initiated and then subcultured on MS medium supplemented with 0.5 mg/l BAP in the light; Hypocotyl-UV: hypocotyl explants were irradiated with UV-C before callus induction and proliferation; Callus- UV: callus which was induced from non-irradiated hypocotyl explants and was irradiated with UV-C. Time (weeks) Betaxanthin content (mg/g FW) control Hypocotyl-UV Callus-UV 0 0.59 ± 0.09b 0.97 ± 0.10a 0.60 ± 0.07b 1 0.59 ± 0.01b 1.01 ± 0.04a 0.95 ± 0.07a 2 0.59 ± 0.04b 1.30 ± 0.16a 1.01± 0.20a 3 0.93 ± 0.14b 1.47 ± 0.12a 1.37± 0.05a 4 0.78 ± 0.03b 1.15 ± 0.04a 1.07 ± 0.04a Values are mean ± SE from 4 replications of analysis. Data marked by same letter in all columns are not significantly different (ANOVA, P< 0.05).

(a) (b)

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Callus induction and influence of UV-C on betalain content in the callus of an amaranth

(c)

Figure 1. Effect of different concentrations of 2,4-D (a), NAA (b) and BAP (c) on fresh weights of calli initiated from hypocotyl explants of Amaranthus cruentus, Celosia argenteavar. plumosaand Gomphrena globosa after culture in the light for 4 weeks. Data (y-axis, mean fresh weights of calli as mg per hypocotyl explant) are from 6 replications (6 jars each containing one hypocotyl explant) and those assigned the same letter indicate the mean fresh weights are not significantly different (P< 0.05, Duncan’s multiple range test).

3. Results Effects of PGRs on callus induction and proliferation The hypocotyls of 7-day-old seedlings of Amaranthus cruentus, Celosia argenteavar. plumosa and Gomphrena globosa were red. When the hypocotyl explants were cultured on MS medium containing 0, 0.5, 1 and 2 mg/l 2,4-D, or NAA or BAP, a variety of responses was observed. No callus was formed in the hypocotyl explants of the three plant species cultured for 4 weeks on PGR-free medium (Figs. 1a to c). Also, the hypocotyl explants of C. argenteavar. plumosadid not form callus on the media supplemented with the different concentrations of 2,4-D (Fig. 1a). The fresh weights of the G. globosacalli formed after four weeks were higher than those of A. cruentus at each of the three concentrations (0.5-2 mg/l) of 2,4-D used. The fresh weight the G. globosacallus induced by 0.5 mg/l 2,4-D was the highest (937.6 mg per explant) compared to the other two concentrations of this PGR (Fig. 1a). In contrast, there were no significant differences in the fresh weights of calli formed in the hypocotyl explants of A. cruentus in response to 0.5 to 2 mg/l 2,4-D. The hypocotyl explants of all three plant species formed calli on NAA-containing medium (Fig. 1b). After the hypocotyl explants of C. argenteavar. plumosa were cultured for 4 weeks, the fresh weight of the callus formed on medium supplemented with 1 or 2 mg/l NAA was higher than on medium supplemented with 0.5 mg/l NAA. Among the NAA concentrations used, the highest callus fresh weight of G. globosaand A. cruentus was on media containing 1 and 2 mg/l of this PGR, respectively. The hypocotyl explants of G. globosa did not form callus in response to the presence of BAP in the medium (Fig. 1c). There was no significant difference in the fresh weights of the calli (about 320 mg per explant) formed on the hypocotyl explants of C. argentea var. plumosa in response to the three concentrations of BAP (0.5 to 2 mg/l). The fresh weights of C. argentea var. plumosacalli were about 6-fold higher than those of A. cruentus. The fresh weight of A. cruentus callus was the highest when its hypocotyl explants were cultured on medium supplemented with 0.5 mg/l BAP compared to the other two concentrations (1 and 2 mg/l) of this PGR. A. cruentus callus induced on BAP-supplemented media was friable and colourless. Besides, all calli of the three plant species formed on NAA-containing media as well as those formed by both G. globosa and A. cruentus on the media supplemented with 2,4-D were also

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KITTI BODHIPADMA, SOMPOCH NOICHINDA, PHETCHARAT WACHIRABONGKOTH, ANGKANA THONGRUANG, KORAVISD NATHALANG, YOSSAPOL PALAPOL, DAVID W. M. LEUNG friable and colourless. Only C. argentea var. plumosa hypocotyl explants cultured on media supplemented with BAP formed red compact callus. During subculture, the fresh weights of the red compact callus of on the three BAP- supplemented mediaincreased continuously (Table 1). The MS medium supplemented with 0.5 mg/l BAP showed the largest increase (about 4 fold) in callus fresh weight after four weeks of subculture (Table 1).

Effects of UV-C on betacyanin and betaxanthin levels At the beginning of subculture, the betacyanin level of the red callus of C. argentea var. plumosa without any prior UV irradiation treatment (control) was the same as that was irradiated immediately before the onset of subculture (Table 2). In contrast, the level of betacyanin inthe callus initiated from hypocotyl explant irradiated with UV-C was higher than the other two treatments at the onset of callus subculture. After 4 weeks of subculture, the betacyanin content in the control callus almost doubled while it increased by more than two times in the two UV treatments (Table 2). The net result of the changes in the betacyanin contents in the callus of the three treatments after 4 weeks of subculture was that the control had the lowest level of betacyanin (0.7 mg/g FW) while the betacyanin contents in the two UV treatments were not significantly different (P<0.05) and were about 70% higher than the control (Table 2). At the beginning of subculture, like the findings about the betacyanin contents in the red calliC. argentea var. plumosa, the control and the callus irradiated immediately before the start of subculture had the same betaxanthin contents while that in the callus derived from UV-irradiated hypocotyl explants was higher (Table 3). However, after 3 weeks of subculture, the control had the lowest betaxanthin content while the calli in the other two UV treatments had the same betaxanthin contents (P<0.05). However, in each of the treatments the increase in betaxanthin contents during callus subculure was less than one fold and was smaller than the increase in the betacyanin contents (Tables 2 and 3). After 4 weeks of subculture, the betaxanthin contents decreased in the calli of all three treatments.

4. Discussion There have been reports on callus induction in Amaranthaceae from leaf disc, hypocotyl and stem explants in the past three decades [18-21]. In the present research, hypocotyl explants of Amaranthus cruentus, Celosia argenteavar. plumosa and Gomphrena globosa exhibited different responses to several types and concentrations of PGRs. BAP and 2,4-D applied singly were not effective for callogenesis in G. globosa andC. argenteavar. plumosa hypocotyl explants, respectively. In contrast, applied NAA was effective for callus induction in all three plant species (Figure 1a-c). Previously, it has been reported that callus formation in hypocotyl explants of A. caudatus, A. cruentus, A. hybridus and A. hypochondriacus required the various combinations of NAA plus BAP or 2,4-D plus kinetin [20], whereas 4 mg/l 2,4-D plus 10% coconut water was used to induce callus of G. globosa [22]. Furthermore, like the hypocotyls of Red Agati seedlings [23] those of A. cruentus, C. argenteavar. plumosa and G. globosa seedlings were also red, suggesting that these could be a good source of red pigments. Interestingly, it was found that production of red pigments did not always accompany callus growth and development from the red hypocotyls of the plants studied here. Only on BAP-supplemented medium, red callus was initiated from the red hypocotyl explants of C. argenteavar. plumosa.

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Callus induction and influence of UV-C on betalain content in the callus of an amaranth

Betacyanins and betaxanthins, the red-violet and yellow-orange pigments, respectively, were the two main groups of nitrogen-containing colorants collectively called betalains. Generally, they are found in plant storage roots, flowers and fruits [9, 24]. However, the present results showed that callus derived from hypocotyl explants of Celosia argenteavar. plumosa could also be an alternative source of these natural colourants. The successful production of betalains via callus of many kinds of plants has been shown before in other studies but most of them used auxin alone or in combination with cytokinin to induce callus [1, 12, 25, 26]. For example, approximately 50 or 80% of the Mammillaria candidacalli were pigmented in media supplemented with auxin and cytokinin or auxin alone, respectively. The pigmentation in the M. candida callus was found to be a modified betaxanthin [27]. Normally, biosynthesis of betalains could be influenced by physical and chemical factors such as temperature, light and PGRs. In plant tissue cultures, cytokininsare able to enhance or reduce the content of betacyanins [27, 28]. For Celosia argenteavar. plumosa callus, BAP alone was enough to induce callus producing these natural pigments. A low concentration (0.5 mg/l) of BAP seemed to be able to promote the accumulation of betalains in C. argenteavar. plumosa callus during subculture. The result showed that Celosia argenteavar. plumosa callus was able to respond to a single 15-min UV-C irradiation treatment before callus subculture as betalain production was enhanced after 4 weeks of subculture. UV-C irradiation of callus of tea plant and jatropha has also been found to influence the production of phenolics and flavonoids in callus cultures of these plants [29, 30]. The mechanism of UV-C irradiation influencing biochemical changes in callus cultures remains to be investigated. Likewise, the hypocotyl explant of C. argenteavar. plumosawas able to respond to the UV irradiation treatment as the red callus initiated from the explant had higher levels of betacyanin and betaxanthin than callus initiated from the non-irradiated explants. It should be noted that previous studies determined biochemical changes in callus within a couple of days following UV irradiation treatment and the assessment of the long-term impact of the treatment on callus culture was seldom carried out (31-34). After the callus of C. argenteavar. plumosa had been subcultured for 4 weeks, the promotive effect of the UV irradiation treatment on betalain production appeared to be sustained during subculture. It seems intriguing and worthwhile for further research to investigate the precise nature of the mechanism of the promotive effect of the treatment. In conclusion, among the three betalain-producing plants only the red hypocotyl explants of Celosia argenteavar. plumosa produced red compact callus on basal MS medium supplemented with BAP. Furthermore, UV-C irradiation treatment applied both before or after callus induction was found to enhance the production of both betacyanin and betaxanthin in the red callus of C. argenteavar. plumosa.

5. Acknowledgements The authors are grateful for the financial support (number 5544101) for this research from the Faculty of Applied Science, King Mongkut’s University of Technology North Bangkok.

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KITTI BODHIPADMA, SOMPOCH NOICHINDA, PHETCHARAT WACHIRABONGKOTH, ANGKANA THONGRUANG, KORAVISD NATHALANG, YOSSAPOL PALAPOL, DAVID W. M. LEUNG

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Callus induction and influence of UV-C on betalain content in the callus of an amaranth

27. M.S. SANTOS-DÍAZ, Y. VELÁSQUEZ-GARCÍA, M.M. GONZÁLEZ-CHÁVEZ. Pigment production by callus of Mammillaria candidaScheidweiler (Cactaceae). Agrociencia, 39: 619-626 (2005). 28. S.-Z. ZHAO, H.-Z. SUN, Y. GAO, N. SUI, B.-S. WANG. Growth regulator-induced betacyanin accumulation and dopa-4,5-dioxygenase (DODA) gene expression in euhalophyteSuaeda salsacalli. In Vitro Cellular & Developmental Biology–Plant,47: 391-398 (2011). 29. N.V. ZAGOSKINA, G.A. DUBRAVINA, A.K. ALYAVINA, E.A. GONCHARUK. Effect of ultraviolet (UV-B) radiation on the formation and localization of phenolic compounds in tea plant callus cultures. Russian Journal of Plant Physiology, 50: 270-275 (2003). 30. E.M. ALVERO-BASCOS, L.B. UNGSON. Ultraviolet-B (UV-B) radiation as an elicitor of flavonoid production in callus cultures of jatropha (Jatropha curcasL.). Philippine Agricultural Scientist, 95: 335- 343 (2012). 31. W. LIU, C.Y. LIU, C.X. YANG, L.J. WANG, S.H. LI. Effect of grape genotype and tissue type on callus growth and production of resveratrols and their piceids after UV-C irradiation. Food Chemistry, 122: 475-481 (2010). 32. E.A. GUENTER, O.A. KAPUSTINA, O.V. POPEYKO, Y.S. OVODOV. Influence of ultraviolet-C on the compositions of cell-wall polysaccharides and carbohydrase activities of Silene vulgaris callus. Carbohydrate Research, 342: 182-189 (2007). 33. M. ZACCHINI, M. DE AGAZIO. Spread of oxidative damage and antioxidative response through cell layers of tobacco callus after UV-C treatment. Plant Physiology and Biochemistry, 42: 445-450 (2004). 34. F. GHANATI, F. KHATAMI. Polyphenols and their antioxidant activity in callus-derived Malvaneglecta cells under UV-B and UV-C irradiation. Planta Medica, 77: 1286-1286 (2011).

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