1. Strategies Applied for the Production of Anthocyanins in Cell and Organ Cultures
In many species, adventitious root, callus, cell, and hairy root cultures have been established; strategies for biomass and anthocyanin production have been applied, including line/clone improvement or callus/cell/organ lines selection, culture medium selection, optimization of nutrient medium, phytohormones, and optimization of physical factors like light, temperature, hydrogen ion concentration (pH), precursors, and elicitors. In the section that follows, these strategies have been explained with appropriate examples.
2. Selection of Cell Lines
It has been shown that the capacity of plant cells to produce secondary metabolites varies significantly. The majority of cell culture research begins with the selection of a high-quality cell line for the accumulation of secondary metabolites
[70][1]. To develop high anthocyanin-yielding cell lines, for instance, cell line selection for
Euphorbia milli was done over numerous callus subcultures, choosing cells with increased anthocyanin content in each subculture
[22][2]. Two cultivars of grapes,
Vitis hybrid Baily Alicant A and
V. vinifera cv. Gamy Freaux, have been routinely utilized to establish cell suspension cultures
[71][3]. Excellent results have been obtained once again through the careful selection of cell clusters over repeated sub-cultures of higher-performing cells
[71,72][3][4]. A further illustration of the significance of effective lines is provided by the hairy root cultures of black carrots. Barba-Espin et al.
[41][5] used wild-type
Rhizobium rhizogenes to produce 93 lines of hairy roots in black carrots. Three fast-growing hairy root lines were chosen, two of which were from root explants (NB-R and 43-R lines) and one from a hypocotyl explant (43-H line). They showed that, in comparison to the original carrot line, the chosen lines produced 25–30 times more biomass and nine distinct anthocyanins.
3. Optimization of Nutrient Medium
Establishing cell and organ cultures for the synthesis of plant secondary metabolites requires careful screening and the selection of appropriate media
[70][1]. Different media formulations, with varying modifications, have been tested for establishing cell and organ cultures in various species for the production of anthocyanin. These include Gamborg’s B5 medium
[73][6], Linsmaier and Skoog or LS medium
[74][7], Murashige and Skoog or MS medium
[75][8], Shenk and Hildebrand or SH medium
[76][9], and Woody plant medium or WPM
[77][10]. In general, cell suspension cultures of
Angelica archangelica,
Aralia cordata,
Cleome rosea,
Daucus carota,
Melostoma malabathricum,
Oxalis linearis,
Panax sikkimensis, and
Prunus cersus could be established using MS media. It was discovered that LS medium was appropriate for cultivating cell cultures of
Perilla frutesens and
Fragaria annanasa; B5 medium was found to be appropriate for
Vaccinium macrocorpon and
Vitis vinifera; and WPM medium was found to be beneficial for
Ajuga pyramidalis cell cultures. The physiological state of the plant species and the kind of culture determine whether a certain medium is appropriate for a given species
[78,79,80,81][11][12][13][14]. When Narayan et al.
[39][15] tested B5, LS, MS, and SH media for their ability to grow
Daucus carota cell cultures, they found that MS medium produced the most biomass and anthocyanin synthesis, while SH medium supported biomass and inhibited anthocyanin synthesis, and other media had lower levels of both biomass and anthocyanin. In hairy root cultures of carrots, Barba-Espin et al.
[41][5] investigated the effects of 1/4, 1/2, and full-strength MS medium on biomass and anthocyanin accumulation. They found that, while full and 1/2 MS were beneficial for biomass accumulation, 1/2 MS was responsible for 4- to 6-fold higher anthocyanin production in various hairy root lines than full MS. They recommended a 1/2 MS medium for the accumulation of biomass and anthocyanin.
4. Influence of Carbon, Nitrogen, and Phosphorous
Optimization of medium, especially with respect to carbon, nitrogen, and phosphorous, has shown a significant impact on the growth of cultured cells and anthocyanin synthesis. The type and concentration of sugars in the medium have demonstrated a profound influence on the growth and synthesis of anthocyanins in cell cultures of varied species. It was demonstrated with grape cell cultures that simple sugars, such as glucose, galactose, and sucrose, or metabolizable sugars support the growth of the cells and accumulation of biomass, whereas non-metabolizable sugars, such as mannitol, are responsible for osmotic stress, which triggers the accumulation of anthocyanins
[82,83][16][17]. Rajendran et al.
[84][18] subjected carrot callus cultures to high concentrations of both sucrose and mannitol and showed that such conditions resulted in an increase in anthocyanin production because of enhanced osmotic conditions. Such carbon effects have been also demonstrated in
Ajuga pyramidalis [25][19],
Cleome rosea [30][20], and
Rosa hybrida [58][21]. In more recent studies, Dai et al.
[85][22] displayed the effect of sugars (glucose, fructose, and sucrose) on in vitro cultured grape berries (in vitro culturing of intact detached grape berries that were grown in greenhouse conditions); their work showed that glucose, fructose, and sucrose increased anthocyanin accumulation, with glucose and fructose being more effective than sucrose. Through molecular analysis, Dai et al.
[85][22] illustrated that sugar induced enhanced anthocyanin accumulation in in vitro-grown grape berries, which resulted from altered expression of regulatory and structural genes, including
CHS,
CHI,
F3′H,
F3H,
DFR,
LAR,
LDOX, and
ANR.
Plant cell culture medium consists of both nitrate and ammonium forms of nitrogen, and the concentration of these two types of nitrogen has been shown to have profound influence on the growth of biomass and anthocyanin synthesis. It has been shown that the reduction of the ammonium form of nitrogen in the medium and enhancement of nitrate nitrogen favored cell growth and anthocyanin accumulation in many types of cell cultures. The concentration of total nitrogen is 60 mM, and the ratio of NH
4+ and NO
3− is 1: 2 in MS medium; when the ratio of NH
4+ to NO
3- was varied from 1:1 to 1:32, keeping nitrogen concentration at 60 mM, there was increased production of anthocyanin (6.8% increment) in carrot cell suspension cultures
[37][23]. However, they recorded optimal cell growth with the medium containing a 1:2 ratio of NH
4+ to NO
3−; therefore, this medium was responsible for a reduction in anthocyanin production of 3.5%. It was reported that cell growth and anthocyanin production was maximized when the ratio of NH
4+ to NO
3− was 1:1 at 60 mM in grape
[86][24], 1:16 at 30 mM in Christ plant
[87][25], 2:28 at 30 mM in strawberry
[43][26], and 1:16 at 30 mM in spikenard
[88][27] cell cultures. These results indicate that the reduction of ammonium nitrogen and increment in nitrate nitrogen favors anthocyanin synthesis in cell cultures of several plants. Another contrasting study by Hirasuna et al.
[60][28] showed that the limitation of overall nitrogen concentration in the culture medium and the increment in sugar levels resulted in a ‘switch-like’ (rather than gradual) enhancement of anthocyanin production in grape cell cultures, which may be equivalent to the removal of an inhibitory effect. The possible explanation given for this short phenomenon is in line with the result which stated that elevated sugars are responsible for increment osmotic potential and concomitant reduction in cell division; this makes nutrient resources more available for secondary metabolism, leading to enhanced biosynthesis of anthocyanin. In contrast, the opposite situation is true for cell growth when higher concentrations of nitrogen levels and appropriate concentration of sugars. Recent studies by Saad et al.
[40][29] have also shown a similar phenomenon in carrot cell suspension cultures. They demonstrated that an altered concentration of NH
4NO
3 and KNO
3 (20:37.6 mM) of MS medium affected the transcription levels of anthocyanin biosynthetic genes, including
PAL,
4CL,
CHS,
CHI,
LDOX, and
UFGT, which is accountable for the increased concentration of anthocyanin content.
Another element that has been demonstrated to have a significant impact on the synthesis of anthocyanins in
Vitis vinifera [63][30] and
Daucus carota [84][18] is the phosphorus level in the cell culture medium. In
Vitis vinifera cell cultures, phosphate levels were reduced from 1.1 mM to 0.25 mM, and without phosphorus. This resulted in increased anthocyanin synthesis by 32% and 46%, respectively
[63][30]. During the culture period, they observed a simultaneous rise in dihydroflavonol reductase activity. Yin et al.
[69][31] investigated the impact of phosphate deficiency on the biosynthesis of anthocyanins in
Vitis vinifera cv. Baily Alicante in a different study. Increases in the expression of transcription factor-encoding gene
VvMybA1 and the flavonoid 3-O-glucosyl transferase (
UFGT) gene are implicated in the pathway leading to anthocyanin production.
5. Influence of Plant Growth Regulators
Plant cell cultures are normally supplemented with varied growth regulators, including auxins such as 2,4-D, IAA, IBA, and NAA, and cytokinins, such as BAP/BA, KN, 2-iP. In several cases, a combination of several auxins with cytokinins has been tested and used efficiently to increase the cell biomass and anthocyanin production. Less frequently, researchers have tested the impact of gibberellic acid (GA
3) and abscisic acid (ABA) in cell cultures of some species. Overall comparative analysis of the impact of growth regulators reveals that the effect of auxins and cytokinins varies from species to species. Among the varied auxins tested, 2,4-D showed promotive effects at lower concentrations, triggering cell growth; however, at elevated concentrations it was found to be inhibitory for anthocyanin production. In carrot (
Daucus carota cv. Kurodagosun) cell suspension cultures, Ozeki and Komamine
[33][32] initially demonstrated that anthocyanin formation was induced by transferring the cells from 2,4-D containing medium to 2,4-D lacking medium. In subsequent studies, Ozeki et al.
[89][33] showed that the carrot cell suspension involved in activities of phenyl ammonia-lyase (PAL), chalcone synthase (CHS), and chalcone-flavanone isomerase (CHFI) activities decreased when they were transferred to the cells from 2,4-D containing medium to 2,4-D lacking medium. Liu et al.
[28][34] demonstrated the function of various auxin concentrations (0, 0.2, 0.4, 2.2, 9, 18, and 27 µM) in transgenic
Arabidopsis thaliana, including IAA, NAA, and 2,4-D. Among transgenic
Arabidopsis thaliana, these auxins variably regulated the expression of genes involved in anthocyanin biosynthesis, including four pathway genes (
PAL1,
CHS,
DFR, and
ANS) and six transcription factors (
TTG1,
EGL3,
MYBL2,
TT8,
GL3, and
PAP1).
Narayan et al.
[39][15] studied the influence of 2,4-D, IAA, and NAA at different levels and recorded the decreased anthocyanin productivity with the increase in 2,4-D levels, and among the three auxins tested, they showed maximum biomass as well as anthocyanin production only when IAA was present in the medium. In addition, Narayan et al.
[39][15] tested the influence of BAP, KN, and 2-iP (0.1–0.4 mg/L) in combination with IAA (2 mg/L), and again, a low level of KN (0.2 mg/L) showed the highest productivity of anthocyanin. Cytokinin’s effect on the gene expression involved in the process of anthocyanin biosynthesis, including
PAL,
CHS,
CHI, and
DFR genes, has been demonstrated by Deikman and Hammer
[90][35]. The addition of KN encouraged the growth of biomass, whereas the substitution of BAP for KN reduced the formation of anthocyanins. In addition to auxins and cytokinins, Gagne et al.
[65][36] have demonstrated the impact of the growth regulator ABA on the formation of anthocyanins in grape cell cultures. Research revealed that the expression of anthocyanin biosynthesis genes, such as
PAL,
C4H,
CHI1, and
CHI2, could be induced when ABA was supplemented in the medium. The findings presented above imply that choosing the right combination and concentration of plant growth regulators is essential for producing anthocyanins and cell biomass in plant cell cultures.
6. Influence of Light, Temperature, and Medium pH
Varied factors, including light, temperature, and medium pH, are reported to be useful components that should be optimized before using the cultures to scale up the process, and these factors influence both biomass and secondary metabolite accumulation in plant cell and organ cultures
[70,78][1][11]. Fluorescent light, when applied to callus or cell suspension cultures of carrot
[91][37], grape
[24][38], strawberry
[92][39], perilla
[53][40], senduduk
[50][41], and cranberry
[59][42], demonstrated a stimulatory effect on biosynthesis and accumulation of anthocyanins. The positive effect of light in relation to PAL activity has been shown by Takeda et al.
[91][37]. The influence of UV-A, UB-B, and UV-C lights has been demonstrated in grape berries; their effect is correlated with the stimulation of expression of the structural genes that encode the enzymes in the shikimate pathway
[93][43].
Studies on
Perilla furtescens [53][40], and
Melastoma malbarthricum [50][41] have examined the effects of temperature regimes during in vitro cell cultures. Generally, relatively higher temperature levels (25 to 30 °C) favored cell growth and biomass accumulation, while relatively lower temperatures (20 °C) facilitated the synthesis of anthocyanins. Zhang et al.
[94][44] used a two-stage culture strategy, maintaining the cultures at 30 °C for three days before moving them to 20 °C. This allowed them to achieve an optimal anthocyanin content of 270 g/L, which was higher than what they would have obtained with a constant temperature treatment. Research conducted by Yamane et al.
[95][45] on the anthocyanin accumulation in berries of the
Vitis hybrid (
V. labrusca ×
V. vinifera) revealed that 20 °C is preferred above 30 °C for a higher anthocyanin accumulation. Although the exact mechanism underlying the temperature effect is yet unknown, Azuma et al.
[96][46] demonstrated the differential expression of MYB-related transcription factors that are temperature-regulated using qRT-PCR study. Additional research is required in this area of study.
The pH or hydrogen ion concentration of the culture media is another crucial element that promotes cell growth and metabolite accumulation. Extreme pH values should be avoided; generally, cells growing in the medium may absorb nutrients efficiently and participate in growth, development, and metabolism at a pH of 5.8
[70][1]. Numerous groups have conducted research on the effects of medium pH on cell proliferation and anthocyanin production. In
Vitis hybrid Baily Alicant A, Suzuki et al.
[97][47] examined cell growth, biomass accumulation, and anthocyanin synthesis in media with varying pH levels, ranging from 4.5 to 8.5. They found that the medium with a pH of 4.5 produced better cell growth and anthocyanin synthesis. Iercan and Nedelea
[98][48] observed a comparable phenomenon in callus cultures of
Vitis vinifera cultivars. The callus cultures of
Feteasca neagra with the highest anthocyanin content (13.5 mg/g FW) were found on the medium with pH = 4.5, whereas the medium with pH = 7.5 revealed 3.3 mg/g FW of anthocyanin. Hagendoorn et al.
[99][49] showed that, in a number of plant species, such as
Morinda citrifolia,
Petunia hybrida, and
Linum flavum, cytoplasmic acidification promoted the formation of secondary metabolites. They demonstrated a rise in PAL activity in response to the culture medium’s cytoplasmic acidification. Numerous studies have shown that higher medium sugar concentrations and nitrate-to-ammonium ratios promote the accumulation of anthocyanins in cell cultures of different species. These characteristics are also responsible for the medium’s pH reduction and the acidification of cultured cells.
7. Elicitation
Elicitors are molecules of biotic and abiotic origin or physical stimuli, such as pulsed electric field or UV irradiation, that can trigger the biosynthesis of secondary metabolites in plant cell and organ cultures
[100][50]. Elicitors such as methyl jasmonate, jasmonic acid, and salicylic acid have been used efficiently in plant in-vitro cultures to activate the biosynthesis of anthocyanins. The extracts of bacterial and fungal origin; insect saliva/varied components of insect saliva; complex polysaccharides, such as chitosan, pectin, alginate, and cyclodextrin; high concentration varied salts, such as CaCl
2, MnSO
4, ZnSO
4, CoCl
2, FeSO
4, VoSO
4, CuSO
4, NH
4NO
3, and KnO
3; ethephon (ethylene producer); UV irradiation; and pulsed electric field stimulus have been tested as elicitors in cell and organ cultures to enhance the anthocyanin biosynthetic pathway.
Treatment of cell suspension cultures of
Vitis vinifera with methyl jasmonate (MeJA) has triggered a 2.9 to 4.1-fold increment in anthocyanin content
[64,101][51][52]. Similarly, MeJA has been shown to enhance the anthocyanin accumulation in callus cultures of
Malus sieversii [49][53]. However, the efficiency of the elicitation process depends on the plant material, culture conditions, contact time, and elicitor concentration. In another study, Qu et al.
[64][51] tested the combined effect of precursor feeding (phenylalanine) treatment with MeJA treatment in grape cell cultures, and they achieved a 4.6-fold increment in anthocyanin accumulation with the addition of 5 mg/L phenylalanine and 50 mg/L MeJA. Sun et al.
[49][53] showed that expression of anthocyanin regulatory (
MdMYB3,
MdMYBB9, and
MdMYB10) and structural (
MdCHS,
MdDFR,
MdF3H, and
MdUFGT) genes increased in
Malus sieversii in response to MeJA elicitation. In addition, Wang et al.
[102][54] demonstrated the upregulation of
MdMYB24L gene in response to MeJA treatment in apple cell cultures, which positively regulates the transcription of
MdDFR and
MdUGFGT genes. The molecular mechanism of JA elicitation is also in line with MeJA action, and Shan et al.
[103][55] deciphered the role of F-box protein CO11 in regulating the transcription factors
PAP1,
PAP2, and
GL3, which were responsible for the expression of anthocyanin biosynthetic genes
DFR,
LDOX, and
UF3GR in
Arabidopsis thaliana.