Effects of Growth Regulators on Production of Anthocyanins in Callus Cultures of Angelica archangelica

Abstract

Anthocyanins are a group of secondary metabolites whose health benefits have been demonstrated in many epidemiological and experimental studies indicating their potential application for the prevention of cardiovascular and neurodegenerative diseases. Plant tissue cultures offer a valuable tool for production of these compounds as an alternative to their isolation from natural sources. Formation of secondary metabolites in vitro depends on many factors. In this work, effects of 4 auxins (2,4-dichlorophenoxyacetic acid, 1-naphthaleneacetic acid, indole-3-acetic acid, and indole-3-butyric acid) and 2 cytokinins (benzylaminopurine and kinetin) at 3 different concentrations (0.1, 1, and 10 mg/L) on cell growth and anthocyanin accumulation in dark-grown callus cultures of Angelica archangelica L. were investigated. The highest biomass and anthocyanin contents were obtained with 1 mg/L benzylaminopurine. Among the auxins, the best results were achieved with either 2,4-dichlorophenoxyacetic acid or 1-naphthaleneacetic acid at 0.1 and 1 mg/L. However, auxins were less effective than cytokinins for growth and anthocyanin accumulation in angelica callus cultures. The influence of auxins and cytokinins on biomass proliferation and anthocyanin production in various plant tissue cultures is discussed.

Plants synthesize a large number of secondary metabolites important for the pharmaceutical, food, cosmetic, and agricultural industries. ^1,2 Most of these plant constituents are difficult to obtain at an industrial level by either chemical synthesis or by microbial cells due to complex chemical structures and complicated biosynthetic pathways. Plant tissue culture technology offers a valuable tool to produce compounds of interest in a desired amount and in an eco-friendly way. ² Anthocyanins are water-soluble phenolic secondary metabolites responsible for a range of red to purple colors in the various parts of plants. ³ Health benefits of dietary anthocyanins have been demonstrated in many epidemiological and experimental studies. For example, the relationship between anthocyanin intake and the reduced risk of obesity, diabetes, and atherosclerosis has been documented. In addition, a neuroprotective activity of anthocyanins for the improvement of cognitive, memory, and motor performance has been suggested, indicating their potential application for the prevention of cardiovascular and neurodegenerative diseases. ⁴ Dark-grown anthocyanin producing callus cultures of Angelica archangelica were established for investigation of conditions influencing in vitro anthocyanin production. ⁵ The culture growth, as well as secondary metabolite formation, depends on a number of factors, among others, on the type and level of plant growth regulators in the nutrient medium. ^1,6 The aim of this work was to study the influence of auxins and cytokinins on cell proliferation and anthocyanin accumulation in angelica callus cultures.

Effects of 4 auxins (2,4-dichlorophenoxyacetic acid [2,4-D], 1-naphthaleneacetic acid [NAA], indole-3-acetic acid [IAA], and indole-3-butyric acid [IBA]) at 3 concentrations (0.1, 1, and 10 mg/L) on culture growth (fresh and dry biomass), and anthocyanin production are summarized in Table 1. The best growth and anthocyanin accumulation in angelica callus cultures were achieved with either 2,4-D or NAA at concentrations of 0.1 and 1 mg/L. 2,4-D or NAA at 10 mg/L were less suitable than those at 0.1 and 1 mg/L—the fresh and dry biomass, and anthocyanins were reduced by approximately 50%, 40%, and 25 - 45%. IAA and IBA at all tested concentrations were not as effective as 2,4-D and NAA at 0.1 and 1 mg/L both for callus proliferation and anthocyanin production—the fresh and dry biomass, and anthocyanin levels declined by about 50%, 40%, and 20 - 40%.

Table 1

Effects of Auxins on Culture Growth and Anthocyanin Accumulation in Angelica archangelica Callus Cultures.

Auxin concentration(mg/L)		Fresh weight(g)	Dry weight(mg)	Anthocyanin content(mg/g dry weight)
2,4-dichlorophenoxyacetic acid	0.1 1 10	3.15 ± 0.59a 3.05 ± 0.30a 1.34 ± 0.15b	154 ± 19a 155 ± 28a 83 ± 6b	5.33 ± 0.59ab 5.25 ± 0.55ac 4.00 ± 0.15d
1-naphthaleneacetic acid	0.1 1 10	3.18 ± 0.39a 3.18 ± 0.50a 1.64 ± 0.21cd	162 ± 12a 161 ± 20a 100 ± 11cd	6.53 ± 1.06a 6.16 ± 0.50a 3.57 ± 0.27e
Indole-3-acetic acid	0.1 1 10	1.58 ± 0.24ce 1.46 ± 0.21be 1.50 ± 0.20def	103 ± 12de 96 ± 11cef 99 ± 13ce	4.35 ± 0.59bcef  4.43 ± 0.30f  4.23 ± 0.35f
Indole-3-butyric acid	0.1 1 10	1.36 ± 0.20bf 1.45 ± 0.22be 1.43 ± 0.17be	90 ± 11f 94 ± 12cf 93 ± 10cf	3.89 ± 0.57ef 4.32 ± 0.49f 4.63 ± 0.44bcf

Each value represents mean ± standard deviation. The values denoted by the same letters within columns are not significantly different, P < 0.05.

Requirements of tissue cultures on auxins for ensuring growth and anthocyanin biosynthesis vary with the plant species. A profound difference in growth as well as anthocyanin contents were not observed in callus cultures of Aralia cordata for 2,4-D, NAA, IAA, or IBA at 0.1, 1, and 10 mg/L, except for 10 mg/L 2,4-D, which produced a strong inhibitory effect. ⁷ 2,4-D remarkably enhanced both cell growth and anthocyanin synthesis in Hibiscus sabdariffa callus cultures as compared with either of the other 3 auxins (NAA, IAA, or IBA). ⁸ It is interesting that 2,4-D at a relatively high concentration of 22 mg/L still showed a growth promoting effect, ⁸ even though weaker than at lower concentrations, in contrast to callus cultures of Aralia cordata, ⁷ and in like manner to those of Angelica archangelica (both of them grown at 10 mg/L 2,4-D). 2,4-D was the most effective phytohormone for cell proliferation in Oxalis reclinata callus cultures; the cultures grew poorly with either NAA or IAA and did not survive with IBA. ⁹ On the other hand, 2,4-D lowered anthocyanin levels, compared with either NAA or IAA. ⁹ The biomass increased while anthocyanin accumulation decreased with rising concentrations of either 2,4-D or NAA in callus cultures of Bridelia stipularis, ¹⁰ Oxalis linearis, ¹¹ and Malus sieversii. ¹² 2,4-D or NAA was more suitable than IAA or IBA for growth in suspension cultures of Fragaria ananassa, ¹³ similarly to angelica callus cultures. However, for anthocyanin production, 2,4-D was the most effective; the influence of NAA, IAA, or IBA was comparable and lower than that of 2,4-D. ¹³ 2,4-D at concentrations from 0.02 to 2.2 mg/L promoted much better cell growth than either NAA or IAA, but completely inhibited (already at 0.02 mg/L) anthocyanin biosynthesis in Daucus carota suspension cultures. ¹⁴ Like in angelica callus cultures, 2,4-D or NAA were more favorable than IAA or IBA for anthocyanin production in grape cell cultures. ¹⁵

Effects of 2 cytokinins (benzylaminopurine [BA] and kinetin [K]) at 3 concentrations (0.1, 1, and 10 mg/L) on culture growth and anthocyanin accumulation in angelica callus cultures are shown in Table 2.

Table 2

Effects of Cytokinins on Culture Growth and Anthocyanin Accumulation in Angelica archangelica Callus Cultures.

Cytokinin concentration(mg/L)		Fresh weight(g)	Dry weight(mg)	Anthocyanin content(mg/g dry weight)
6-benzylaminopurine	0.1 1 10	4.77 ± 0.42a 6.15 ± 0.51b 0.56 ± 0.05c	213 ± 13a 231 ± 16b 35 ± 6c	9.03 ± 0.82a 11.22 ± 0.91b 0.99 ± 0.13c
Kinetin	0.1 1 10	3.48 ± 0.55d 5.52 ± 0.70a 0.80 ± 0.10e	171 ± 15d 224 ± 18ab 48 ± 6e	6.21 ± 0.71d 10.13 ± 0.19b 1.25 ± 0.03e

Each value represents mean ± standard deviation. The values denoted by the same letters within columns are not significantly different, P < 0.05.

The best results were obtained with either BA or K at a concentration of 1 mg/L; moreover, BA or K at 1 mg/L were more effective than 2,4-D or NAA at 0.1 and 1 mg/L. With BA or K at 0.1 mg/L, callus fresh and dry weights, and anthocyanin contents declined by 22% and 37%, 8% and 24%, and 20% and 39%, respectively, compared with BA or K at 1 mg/L. BA or K at 10 mg/L failed to promote either cell proliferation or production of anthocyanins in angelica callus cultures. Similarly, at 0.1 and 1 mg/L BA or K well facilitated growth and anthocyanin biosynthesis in callus cultures of Aralia cordata, whereas both were remarkably reduced at 10 mg/L. ⁷ On the other hand, in contrast to angelica callus cultures, growth and production supporting activities of either BA or K were comparable, not higher, to those of the auxins tested (2,4-D, NAA, IAA, or IBA). ⁷ Callus cultures of Bridelia stipularis grew well (although to a slightly lesser degree than with 2,4-D or NAA), but produced no anthocyanins on media supplemented with either BA or K. ¹⁰ BA at 1.8 mg/L exerted a detrimental impact on growth (substantially reduced in comparison with NAA), but a significantly stimulating effect on anthocyanin biosynthesis (higher than NAA) in callus cultures of Oxalis linearis. ¹¹ BA at 1 mg/L supported cell proliferation (much higher than NAA or IAA, and lower than 2,4-D) and anthocyanin accumulation (better than 2,4-D, comparable with IAA, and less than NAA) in Oxalis reclinata callus cultures. ⁹ Contrarily, the cultures were not able to live and produce anthocyanins at all on a medium supplemented with K. ⁹

BA or K (both of them alike) at 0.02 and 0.2 mg/L promoted moderate cell growth and considerable anthocyanin biosynthesis (compared with the auxins 2,4-D, NAA, and IAA), whereas at 2 mg/L both of them completely suppressed (like auxins) in Daucus carota suspension cultures. ¹⁴ The biomass decreased while anthocyanin accumulation increased with rising concentrations of BA or K from 0.02 to 11 mg/L in cell cultures of Haplopappus gracilis. ¹⁶ BA outperformed K in positive influence on both growth and anthocyanin content in Vaccinium pahalae suspension cultures. ¹⁷ In agreement with results from angelica callus cultures, BA at 1 mg/L gave rise to the maximum anthocyanin content and higher BA concentrations decreased it in callus cultures of Malus sieversii. ¹²

It may be noted that the effects of particular auxins or cytokinins alone on cell proliferation and anthocyanin production in angelica callus cultures were not better than those of a 2,4-D and BA combination used in the maintenance medium, except for either BA or K at 1 mg/L when the efficiency was similar to that of 2,4-D with BA. Using that medium, fresh and dry biomass, and anthocyanin content were 5.05 ± 0.60 g, 205 ± 18 mg, and 9.14 ± 1.18 mg/g dry weight, respectively. This corresponds with findings in callus cultures of Aralia cordata where the highest anthocyanin production was achieved with a combination of either NAA or 2,4-D and K (the latter was used in the maintenance medium), in comparison with auxins or cytokinins used separately. ⁷ Combinations of auxins and cytokinins can influence growth and anthocyanin accumulation both positively and negatively. NAA with BA worsened or improved growth in comparison with NAA or BA, respectively, in callus cultures of Oxalis linearis. ¹¹ Anthocyanin contents were similar or substantially stimulated, compared with those on media supplemented with BA or NAA, respectively. ¹¹ In that case, advantageously, adverse effects of BA on cell proliferation and those of NAA on anthocyanin production were mutually counteracted by a combination of both. In another case, in callus cultures of Malus sieversii, BA in the presence of NAA was more beneficial to anthocyanin accumulation than BA alone. ¹² Addition of BA or K to 2,4-D was detrimental to anthocyanin biosynthesis in grape cell cultures. ¹⁵ Combinations of 2,4-D and K enhanced cell growth and, on the other hand, little affected anthocyanin levels in Hibiscus sabdariffa callus cultures, as compared with 2,4-D singly. ⁸

In conclusion, results of this study, as well as literature reports confirm that particular auxins or cytokinins, either alone or in combinations, affect cell growth and anthocyanin accumulation in plant tissue cultures very differently. Their effects are hardly predictable because exact mechanisms underlying regulation of primary and secondary metabolism by auxins and cytokinins are not yet fully known. ^12,18,19 Therefore, a suitable type and concentration of growth regulators have to be determined for each culture experimentally.

Experimental

Plant Material and Culture Conditions

Callus cultures of Angelica archangelica derived from seedlings as described previously ⁵ were maintained on a Murashige and Skoog medium ²⁰ supplemented with 2 mg/L 2,4-dichlorophenoxyacetic acid, 0.4 mg/L 6-benzylaminopurine, and 30 g/L sucrose. The pH of all media was adjusted to 5.7 before autoclaving at 121°C for 15 minutes. The cultures were incubated at 25 ± 1°C in the dark and subcultured every 4 weeks. For testing the effects of various growth regulators, the cultures with an initial inoculum of 500 ± 50 mg were cultured on a Murashige and Skoog medium supplemented with an appropriate auxin or cytokinin at a concentration of 0.1, 1 or 10 mg/L. After 30 days, the cultures were harvested, and callus growth and anthocyanin production were evaluated. Experiments were carried out 3 times with 5 replicates for each treatment.

Measurement of Culture Growth

The harvested calli were weighed for fresh weight determination, and then freeze-dried to obtain dry weight.

Quantitative Determination of Anthocyanins

Anthocyanins were quantified spectrophotometrically. ²¹ The powdered freeze-dried calli were extracted in methanol containing 1% HCl for 14 h (overnight) at 4°C. The extracts were centrifuged at 2500 rpm for 10 minutes. The absorbance of the clear extracts was measured at 535 nm. The anthocyanin content was calculated using a calibration curve of cyanidin-3-glucoside and expressed in mg per 1 g of dry weight.

Statistical Analysis

Data obtained were expressed as means with standard deviations and were statistically analyzed using a one-way analysis of variance, followed by Tukey’s multiple comparison test. Differences at P < 0.05 were regarded as statistically significant.