Table 1.
Successful examples of anthocyanin production from in vitro cell and organ cultures.
| Plant Species | Type of Culture |
Medium Composition |
Strategy Followed | Response | Total Anthocyanin Content |
References |
|---|---|---|---|---|---|---|
| Ajuga pyramidalis Metllica crispa | Cell suspension culture | Woody plant medium supplemented with 2.26 µM 2,4-D and 3.49 µM KN | The effect of different sugars was tested The effect of PGR was evaluated |
Fructose in the medium resulted in the highest FW, whereas, galactose enhanced AP. 2,4-D plus kinetin resulted in higher growth, whereas a combination of IAA or NAA with zeatin resulted in the highest AP |
0.29 mg/treatment | [25] |
| Angelica archangelica | Callus culture | MS medium with 30 g/L sucrose | The effect of PGR was evaluated |
The highest biomass, of 231 mg DW, anthocyanin (11.22 mg/g DW), was achieved on a medium supplemented with 1 mg/L BAP | 11.22 mg/g DW | [26] |
| Arabidopsis thaliana | Growth of seedlings after seed germination | MS medium with 3% sucrose | The effect of nitrogen in the MS medium was tested The effect of light was evaluated |
The combined growth conditions of high light conditions induced the most molecular diversity and transcript levels of PAP1, PAL1, CHS, DFR, and ANS genes | NR | [27] |
| Arabidopsis thaliana | Callus culture | Transformed calli with pap1-D (production of anthocyanin pigmentation 1-Dominant) and wild-type calli were maintained on MS medium with 0.4 mM potassium nitrate (KNO3) and without ammonium nitrate (NH4NO3) | The effect of PGR was tested | 2,4-D, NAA, and IAA control AP by regulating the expression of TT8, GL3, and PAP1 (genes involved in regulating the gene expression) as well as DFR and ANS (genes involved in the anthocyanin biosynthetic pathway) | Anthocyanin content is expressed in relative units | [28] |
| Aralia cordata | Cell suspension culture | Varied media were tested | The effect of different media was tested |
LS medium was good for biomass growth, however, B5 medium was good for AP | 10.3% DW | [29] |
| Cleome rosea | Cell suspension culture | MS medium | The effect of salt strength on the medium was tested The effect of sucrose levels was evaluated |
AP was highest in cell suspensions grown on half-strength MS, 30 g/L sucrose, and 0.45 µM 2,4-D | 20.32 color value/g FW | [30] |
| Daucus carota | Callus culture | Modified MS medium with 0.1 mM 2,4-D and 60 mM sucrose | Selection of high-yielding cell line | Cultures of the smaller size had higher anthocyanin content than those of the larger-size | Anthocyanin content is expressed as relative absorbance units | [31] |
| Daucus carota | Callus culture | Modified MS medium with 0.1 mM 2,4-D and 60 mM sucrose | Selection of high-yielding cell line |
Lines with higher levels of anthocyanin were selected | 1–2% | [32] |
| Daucus carota cv. Kurodagosun | Cell suspension culture | MS medium containing 0.5 µM 2,4-D and 2% sucrose | The effect of 2,4-D was tested |
The activity of varied enzymes involved in AP was suppressed with the addition of 2,4-D | NR | [33] |
| Daucus carota | Cell suspension culture | B5 medium with 0.1 mg/L 2,4-D and 100 mg/L MnSO4 | The effect of different media was tested The effect of different sugars in the medium was tested |
Of the varied media tested B5 medium resulted in the highest AP; Among the various carbohydrates, 20 g/L of galactose concentration resulted in the maximum cell yield and AP Of the varied combinations of sugars tested highest cell volume index and relative AP were observed in 15G:5S at 5% inoculum density |
NR | [34] |
| Dacus carota | Callus culture | MS medium with 2 mg/L 2,4-D, 0.2 mg/L KN, 3% sucrose | The effect of fungal elicitors was evaluated |
Aspergillus flavus extract at the 2.5% level resulted in the accumulation of a 2-fold increase in AP | 20% DW | [35] |
| Daucus carota | Callus culture | MS medium with 2 mg/L 2,4-D, 0.2 mg/L KN, 3% sucrose | The effect of fungal extracts and metal ions were evaluated |
All elicitors showed enhanced AP | NR | [36] |
| Daucus carota cv. Nantes scarlet 104 | Callus culture | MS containing 2 mg/L 2,4-D, 0.2 mg/L KN | The effect of sugars and their concentrations was evaluated The effect of nitrogen levels were tested |
Glucose and sucrose produced 3.5% (dry weight basis) AP, whereas supplementation of 7.5% sucrose to the medium produced the maximum AP (6.5%). Similarly, total nitrogen at 70 mM concentration and a 1:4 ratio of ammonium and nitrate yielded maximum callus growth and best AP | 6.5% DW | [37] |
| Daucus carota var. Nantes scarlet | Callus culture | MS medium with 2 mg/L 2,4-D, 0.5 mg/L KN, 3% sucrose | The effect of MJ, and SA was tested |
With the treatment of 200 µM SA and 0.01 µM MJ, an increment in AP was realized | 0.36% and 0.37% (Control 0.22%) | [38] |
| Daucus carota cv. Nantes scarlet 104 | Callus/cell suspension culture | The effect of MS/LS/B5/SH media was tested | The effect of different media and PGR were tested The effect of temperature was evaluated |
Maximum biomass and AP were with MS medium. Among varied combinations of MS with IAA (4.0 mg/L) and KN (0.4 mg/L) was excellent for biomass and AP in liquid cultures. Of the varied temperature regimes 25 °C was excellent for both biomass and AP |
2–4% | [39] |
| Dacus carota | Cell suspension culture | MS medium containing IAA (11.41 µM) and KN (0.93 µM) | The effect of nitrogen and phosphate levels was evaluated |
The highest AP was obtained in the medium supplemented with 20.0:37.6 mM (NH4NO3:KNO3 ratio) on the 15th day. Similarly, a phosphate level of 0.45 mM in the medium supported the highest fresh cell weight (15.68 g/L) as compared to the culture supplemented with different concentrations of KH2PO4 (0, 0.225, 0.9, 1.8, or 2.7 mM), whereas maximum anthocyanin content was seen for 0.45 mM phosphate, which was 1.63-fold higher than in the control cultures |
3.2 mg/g FW with the optimized nitrogen supplements and 3.66 mg/g FW with the optimized phosphate levels. | [40] |
| Daucus carota ssp. sativus var. atrorubens | Hairy root culture | Quarter, half, and full MS mediums were tested | The effect of medium strength was evaluated |
Half-strength MS was suitable and 6-fold higher AP was accumulated compared to control | 3.03 mg/g DW | [41] |
| Dacus carota var. Atomic red | Cell suspension culture | MS medium containing 9.1 µM IAA and 2.32 µM KN | The effect of salt stress was evaluated |
The maximum AP was recorded in a salt stress medium of 4.33 mg/L FW on day 9 which was two-fold higher than the control | 10.91 mg/L FW | [42] |
| Fragaria ananassa cv. Shikinari | Cell suspension culture | LS or B5 medium with 1 mg/L 2,4-D and 0.1 mg/L BA | The effect of various sugars was evaluated The effect of nitrogen concentrations were tested |
The maximum yield of AP was realized in a modified LS medium containing 5% sucrose, a ratio of NH4+ (2 mM): NO3− (28 mM) | 380 µg/g FW | [43] |
| Fragaria ananassa cv. Shikinari | Cell suspension culture | LS medium supplemented with 3% sucrose, 0.1 mg/L BA, and 0, 0.01 and 1 mg/L 2,4-D | The effect of 2,4-D concentration was evaluated |
Lower 2,4-D (0.1 mg/L) concentrations in the medium limited the cell growth and enhanced AP | 1200 µg/g FW | [44] |
| Fragaira annanassa cv. Shikinari | Cell suspension culture | LS or B5 medium supplemented with 30 g/L sucrose, 1 mg/L 2,4-D and 0.1 mg/L BA | Repeated-batch culture strategy was evaluated on different media |
The average AP was enhanced 1.7- and 1.76-fold by repeated-batch cultures in constant LS and constant B5 medium at a 9-day shift period for 45 days | 0.42–0.52 mg/g FW | [45] |
| Fragaria ananassa cv. Shikinari | Cell suspension culture | LS medium with 1 mg/L 2,4-D, 0.1 mg/L BAP, 0.4 mg/L thiamine hydrochloride | Effect of precursor L-phenylalanine was evaluated |
In repetitive feeding culture, a maximum of 81% higher AP was realized | 58 mg/L | [46] |
| Haplopappus gracilis | Callus culture | Initial cultures were raised on a White medium with 2% sucrose and 10% coconut milk and 0, 0.1, 1.0, and 10 mg/L 2,4-D. During the second stage Sunderland medium with 2% sucrose and 10% coconut milk | The effect of 2,4-D, dark, and light incubation was evaluated |
The light was essential for AP; however, 2,4-D suppressed the AP | 3.9 mg/g DW | [47] |
| Malus sieversii f. niedzwetzkyana | Callus culture | MS medium with 3% sucrose | The effect of PGR levels and combination was evaluated |
Auxin alone with the increment of concentration inhibited AP and molecular analysis showed that anthocyanin regulatory genes (MdMYB10 and MdbHLH3) were dramatically suppressed by 0.6 mg/L 2,4-D. The BAP, TDZ, and nitrogen lower concentration of 2,4-D in combination with cytokinin enhanced the AP | Anthocyanin content is expressed as relative absorbance units | [48] |
| Malus sieversii f. niedzwetzkyana | Callus culture | MS medium with 4 µM BA, 2 µM NAA | The effect of MJ and ABA were tested |
Studied the expression of gene expression and AP: Gene expression indicated that MJ induced expression of MdMYB9, MdMYB10, Md CHS, MdF3H, and MdUFGT genes. ABA inhibited the expression of MdMYB3, MdMYB10, MDF3H, MdDFR, MdDOX, and MdUFGT genes. Overall, 26% increment increase in cyanidin 3-O-galactoside content |
Anthocyanin content is expressed as relative absorbance units | [49] |
| Melostoma malabthricum | Cell suspension culture | MS medium supplemented with 0.25 mg/L BA and 0.5 mg/L NAA | The effect of light intensity was evaluated The temperature levels were tested |
The cultures exposed to light intensity of 301–600 lux depicted the highest biomass and AP; Cultures exposed to 20 °C have accumulated higher biomass and anthocyanin than 26 and 29 °C |
1.62 color value/g FW | [50] |
| Oxalis linearis | Callus culture | MS medium supplemented with 60 mM sucrose | The effect of PGR was tested The effect of photoperiod was evaluated |
Callus growth was stimulated at a concentration of 8–32 µM NAA and 2,4-D. BA and Zeatin inhibited callus growth, whereas IP stimulated callus growth. The optimum AP was with NAA at 8 µM NAA. Sucrose at 60 mM was best for callus growth and 240 mM was good for AP |
NR | [51] |
| Panax sikkimensis | Callus culture | MS medium containing 3% sucrose, 0.01% myoinositol, 0.33 µM thiamine hydrochloride, 2.5 µM pyridoxine hydrochloride, 4.0 µM nicotinic acid, 4.5 µM 2,4-D, and 1.2 µM KN | Cell line selection The effect of photoperiod was evaluated |
The selected line had the highest growth and productivity (2.192 mg/g FW and 6.92% DW); The continuous exposure to light favored both biomass and AP |
2.76 mg/g FW (7.07% DW) | [52] |
| Perilla frutescens | Cell suspension culture | LS medium with 3% sucrose, 1 µM 2,4-D and 1 µM BA | The effect of temperature was evaluated |
At temperatures of 22, 25, and 28 °C, the specific growth rate of cells was 0.21, 0.32, and 0.37 per day, respectively. However, the AP was reduced compared to 25 °C. Therefore, a temperate culture temperature of 25 °C was suggested for the maintenance of cultures | NR | [53] |
| Perilla frutescens | Cell suspension culture | LS medium with 3% sucrose, 1 µM 2,4-D and 1 µM BA | The effect of inoculum density was evaluated |
Optimum AP (3.6 g/L) was reported at an inoculum size of 50 g/L | 117.6 mg/g DW | [54] |
| Perilla frutescens | Cell suspension culture | MS medium supplemented with 0.2 mg/L 2,4-D and 0.5 mg/L BA | The effect of yeast extract was evaluated |
Increment increase in AP (10.2%) with the addition of 1% yeast extract | 10.2% DW | [55] |
| Prunus cerasus cv Amarena Mattarello | Callus culture | MS medium containing 30 g/L sucrose, 1 mg/L NAA, 0.1 mg/L BAP | The effect of JA was evaluated The light effect was tested |
Cultures elicited by 50 µM JA stimulated the accumulation of cyanidin 3-glucoside accumulation When cultures were exposed to light, cyanidin 3-glucoside content was increased from 0.1 to 4.5 mg/100 g FW |
5.3 mg/100 g FW | [56] |
| Raphanus sativus cv Peking Koushin | Adventitious root culture | Half strength MS | The effect of PGR was tested The effect of light was evaluated |
Cultures with IBA IBA-supplemented medium and incubated in light involved in AP than cultures supplemented with NAA and dark incubation | 0.15% DW | [57] |
| Rosa hybrida | Callus culture | B5 medium with 0.2 mg/L 2,4-D | The effect of sucrose concentration was tested The effect of nitrogen levels was evaluated |
The optimum amount of AP (7.20 mg/kg FW and 6.58 mg/kg FW) was recorded on medium supplemented with 5% sucrose and medium devoid of NH4+ respectively | 7.20 mg/kg FW | [58] |
| Vaccinium macrocarpon | Callus culture | Modified B5 medium with 5.7 µM NAA, 0.45 µM 2–4-D, and 2.32 µM KN in the dark at 25 °C | The effect of light was evaluated |
Cultures exposed to light have shown maximum concentration of AP | 140 µg/g FW | [59] |
| Vitis hybrid (Bailey Alicang A) | Cell suspension culture | B5 medium with 2% sucrose and 0.5 mg/L 2,4-D (Maintenance medium) | The combined effect of nitrogen and sucrose was evaluated |
The combined effect of low nitrate and high sucrose synergistically improved AP | 2.9 g/L | [60] |
| Vitis vinifera cv. Gamay Freaux | Cell suspension culture | B5 medium | The combined effect of nitrogen and sucrose was evaluated |
The combined effect was responsible for the increase in anthocyanins, especially peonidin 3-glucoside | NR | [61] |
| Vitis vinifera var. Gamay Freaux | Callus culture | B5 medium with 88 mM sucrose, 250 mg/L casein hydrolysate, 0.54 µM NAA, 0.93 µM KN | Selection of cell line | Cell lines that were accumulating higher levels of peonidin 3-glucoside (line 5.4) and peonidin 3-p-coumaroylglucoside (line 13.1) were selected | 1.02 mg/g FW | [62] |
| Vitis vinifera cv. Gamay Freaux | Cell suspension culture | Modified MS medium with B5 macro-elements, MS micro-elements, 2% sucrose, 0.025% casein hydrolysate, 0.1 mg/L KN and 0.1 mg/L NAA | The effect of phosphate level was tested |
Deprivation of phosphate led to enhanced synthesis of anthocyanin by 32% and 46% in P2 and P3 media, respectively | NR | [63] |
| Vitis vinifera cv. Gamay Freaux var. Teinturier berry | Cell suspension culture | B5 medium with 30 g/L sucrose, 250 mg/L casein hydrolysate, 0.1 mg/L NAA, 0.2 µM KN | The effect of precursor phenylalanine and MJ was evaluated |
Treatment with 5 mg/L phenylalanine, 50 mg/L MJ, and 1 mg/L dextran enhanced the 4.6-fold of AP | 7.09 color value/g DW | [64] |
| Vitis vanifera cv Cabernet Sauvignon | Cell suspension culture | B5 medium supplemented with 20 g/L sucrose, 250 mg/L casein hydrolysate, 0.5 mg/L NAA, 0.12 mg/BA | The effect of ABA was evaluated |
ABA-treated cells exhibited an earlier increase in VvCHI1, VvCHI2, VvC4H, and VvMYBA1 transcripts and AP | NR | [65] |
| Vitis vinifera cv. Gamay Freaux | Cell suspension culture | B5 medium | The effect of indanoyl-isoleucine (In-Ile) was evaluated |
In-Ile was a potent elicitor in stimulating AP and 2.6, 1.8, and 1.9-fold increments in anthocyanin production on 8 d, 10 d, and 12 d after the treatments, respectively | 4.6 mg/g DW | [66] |
| Vitis vinifera cv. Gamay Freaux | Cell suspension culture | B5 medium 0.1 mg/L NAA, 0.2 mg/L KN, 0.25 mg/L casein hydrolysate, 3% sucrose | The effect of elicitors was evaluated |
Chitosan, pectin, and alginate enhanced the production of anthocyanin by 2.5, 2.5, and 2.6-fold, respectively | 4.2 mg/g DW | [67] |
| Vitis vinifera cv. Gamay Freaux | Cell suspension culture | B5 medium with 0.1 mg/L NAA, 0.2 mg/L KN, 0.25 mg/L casein hydrolysate, 3% sucrose | The effect of ethephon and pulsed electric field was evaluated |
The treatment of ethephon resulted in a 2.3-fold increase (1.99 mg/g DW) and 2.3-fold (1.99 mg/g DW) in anthocyanin content, while combined treatment with both ethephon and PEF resulted in 2.5-fold increase (2.2 mg/g DW) in AP | 2.2 mg/g DW | [68] |
| Vitis hybrid Bailey Alicante A | Cell suspension culture | MS medium supplemented with 3% sucrose, 0.23 µM 2–4-D, 1 µM KN, 3 µM thiamine-HCl, 560 µM myo-inositol | The effect of phosphate was evaluated |
Cynidin-3-O-glucoside, peonidin-3-O-gluside, cyanidin-3-O-(6-O″-p-coumaroyl)-glucoside and peonidin-3-O-(6-O″-p-coumaroyl)-glucoside were detected in higher concentration in Pi deprived cells. The transcript levels of (UFGT) and VvmbyA1 were also higher in Pi-deprived cells | NR | [69] |
ABA—Abscisic acid; AP—Anthocyanin production; 2,4-D—2,4-dichlorophenoxy acetic acid; B5—Gamborg B5 medium; BA—benzyladenine; BAP—benzylaminopurine; DW—dry weight; FW—fresh weight; IAA—indole acetic acid; IBA—indole butyric acid; IP—isopentenyl adenine; JA—Jasmonic acid; KN—kinetin; LS—Linsmaier and Skoog medium; MJ—methyl jasmonate; MS—Murashige and Skoog medium; NAA—naphthalene acetic acid; NR-not reported; PGR—Plant growth regulator; SH—Schenk and Hildebrandt medium; TDZ—thidiazuron.