1. Introduction
Research continues to show that many artificial pigments are actually detrimental to our health. According to [1], there is an increasing consumer preference for healthy foods, which has invited considerable demand for the use of anthocyanins as natural colorants, because of their natural pedigree and healthful properties. Anthocyanins are the most widely distributed group of water-soluble plant pigments in nature. They are mainly responsible for the mauve, red, blue, and purple colors in flowers, fruits, leaves, seeds and other organs in most of the flowering plants. The other important class of water-soluble pigments are betalains, which are present only in plants belonging to 13 families of Caryophyllales order [2-5]. An interesting phenomenon is the existence of mutual exclusiveness of anthocyanins and betalains in plant kingdom [3, 5-9]. Recent research demonstrated that simultaneous production of anthocyanins and betalains is possible in cell cultures and seedlings of anthocyanin producing plants by introduction and expression of genes encoding dihydroxyphenylalanine (L-DOPA) dioxygenases in combination with substrate precursor feeding [10]. However, the co-occurrence of both pigments in the same plant species have never been found in nature and the plants which produce anthocyanins never produce betalains and vice versa [6]. The commercial production of anthocyanin pigments is one of the fastest growing segments of the food colorant industry [2, 11]. The only industrial sources for anthocyanin pigments are from whole plant extracts [1], with the most common source being grape skins from the wine industry. According to [1], the demand of natural colorants continues to rise by 5-15% every year and this translated to the sales of anthocyanins isolated from grape skins in 2002, which was estimated to be US$200 million worldwide. The increase in demand for processed foods and high health products has caused the manufacturers to look for alternative sources of colorants with antioxidant properties. One source is the production of anthocyanins through the use of plant cell cultures [2, 12, 13].
Anthocyanins are synthesized via the flavonoid pathway, and they are known to contribute red, blue and purple color to colored grapes, wines and other products [14-18]. Anthocyanins can be used not only as food and beverage additives to obtain attractive natural coloration [19], but also for generating pharmaceutical and cosmetic products. Most researchers are optimistic about utilizing them as bioactive compounds with the consideration that they have the potential to improve human health [1]. Anthocyanins have been implicated in lowering the risk of cardiovascular disease and certain cancers. Dietary anthocyanins can be obtained by humans through the ingestion of fresh colored fruits processed into food and beverages. For instance, the consumption of red grapes and wine is considered vital for bioavailable anthocyanins [20, 21]. To date, most anthocyanin colorants are extracted from grape skins, black carrots, red cabbage, and sweet potato [11]. However, researchers are also exploring the idea of cultivating plant cell cultures for the production of natural colorants. Therefore, there is an interest in improving the quantity and quality of anthocyanins produced in grape cells, and this means that commercially viable systems must be developed to produce anthocyanins in grape cell cultures.
Production of anthocyanins by plant cell cultures is a feasible technology being pursued by industrial and academic interests. Several strategies are being used to enhance anthocyanin biosynthesis in plant cells. This involves a proper selection of the cell strain and optimization of media as well as culture conditions. It is crucial to note that anthocyanins obtained directly from fresh plant materials has limitations such as low metabolite yield, variability, and seasonal availability of raw materials, fresh material losses, inconsistent product quality, and pigment degradation caused by storage and extraction process [22]. Therefore, it is prudent to use
2. Biology and chemistry of anthocyanins as pigments
As colored molecules, anthocyanins play a key role in survival and evolution of flowering plants by attracting pollinators, frugivores and seed dispersers on one hand, and by repelling herbivores and parasites on the other [24-26]. Moreover, anthocyanins execute several important physiological functions in plant cells, and their biosynthesis is strongly induced by biotic and abiotic stress factors. These factors include, light, UV radiation, high or low temperatures, wounding, osmotic stress, nutrient imbalance, ozone exposure, herbivores, microbial and viral attacks. In [24, 27] the major roles of anthocyanins in photoprotection of chloroplasts from photoinhibitory damage have been discussed in details. The authors have also clarified the involvement of anthocyanins in protection from UV-B radiation, as well as how anthocyanins decrease oxidative stress by scavenging free radicals and modulating reactive oxygen signaling cascades. These cascades are responsible for triggering the expression of stress-responsive genes as well as the regulation of plant growth and development [24, 27].
Structurally anthocyanins are substituted glycosides and acylglycosides of 2-phenylbenzopyrilium salts (anthocyanidins). The basic structure of anthocyanidins consist of a chromane ring (C-6 – ring A and C-3 – ring C) bearing a second aromatic ring (C-6 – ring B) in position 2 (Figure 1) [2, 5, 28-30]. The various anthocyanidins differs in number and position of the hydroxyl and /or methyl ether groups attached on 3, 5, 6, 7, 3’, 4’ and/or 5’ positions. Despite the fact that 31 different monomeric anthocyanidins have been identified (including 3-deoxyanthocyanidins, pyranoanthocyanidins and sphagnorubins), 90% of the naturally occurring anthocyanins are based on only six structures (30% on cyanidin
The color of anthocyanidins differs with the number of hydroxyl groups, attached on their molecules (especially those substituted in ring B). With the increase of attached hydroxyl groups, the visible color of entire molecule shift from orange to violet (Figure 2) [2, 5, 29, 30]. Glycosylation of anthocyanidins results to additional reddening of obtained anthocyanins, whereas the presence of aliphatic or aromatic acyl moieties causes no color change or slight blue shift and has significant effect on their stability and solubility [5]. Changes in pH can also cause reversible structural transformations in anthocyanins molecules, which has a dramatic effect on their color (Figure 3) [30-34].
Most of the anthocyanins are
2.1. Anthocyanin biosynthesis in grapes
As a major flavonoid group, anthocyanins are products of phenylpropanoid metabolism of plant cells [28, 29, 38]. Anthocyanins in grapes are synthesized via flavonoid pathway. The biosynthetic pathway can be divided into two sections, the basic flavonoid upstream pathway, which includes early biosynthetic genes (EBGs), and the specific anthocyanin downstream branch, which includes late biosynthetic genes (LBGs) (Figure 5). Studies have shown that the basic flavonoid upstream pathway is restricted in many plants [39, 40, 41, 42] and that large gene families encodes the enzymes that act early in the flavonoid pathway, while the enzymes acting late in the pathway are encoded by single active gene [43]. The flavonoid pathway starts with phenylalanine, produced via shikimate pathway and transformed to 4-coumaroyl-CoA. The key enzyme, chalcone synthase (CHS) produce a naringenin chalcone by condense of one molecule 4-coumaroyl-CoA and three malonyl-CoA molecules (derived from citrate produced by The Krebs cycle) (Figure 5) [44]. In this case, the rings A and C are derived from the acetate pathway, whereas the ring B is derived from shikimate pathway [45]. Currently, there are three genes encoding CHS in grapes:
After this reduction, anthocyanidin synthase (ANS) oxidize leucoanthocyanidins to their corresponding anthocyanidins. Anthocyanidins are inherently unstable under physiological conditions and were immediately glycosylated to anthocyanins by UDP-glucose: Anthocianidin: Flavonoid glucosyltransferase (UFGT) [48]. Anthocyanins, containing methylated anthocyanidins (peonidin
2.2. Anthocyanins storage in grape cells
Once anthocyanins have been produced, they are transported and stored into the cell vacuole. Inside of vacuole, anthocyanins could be connected to specific proteins forming nomembrane intravacuolar bodies, known as anthocyanic vacuolar inclusions (AVI) (Figure 6) [50]. It has been confirmed that AVI plays a critical role in formation of color in flowering plants [50]. Recently, AVI from grape cell suspension were isolated and analyzed [51]. In contradiction with other plants, it was demonstrated that in grape cell suspension AVI consist of complex mix of tannins, anthocyanins (predominantly acylated derivates), proteins and other organic compounds, encased by lipid membrane [32, 51]. It was observed that a strong correlation between the prevalence of AVI structures in grapevine cell suspensions and the increase of their anthocyanin accumulation exist [51]. However, the enhancement of AVI prevalence does affect neither the number of available pigmented cells nor the overall growth rate of suspension cultures. Since AVI plays an important function in the storage and concentration of anthocyanins in cell vacuoles, their perspective role as enhancers of anthocyanin accumulation in grape cell suspensions have been proposed [51].
2.3. Characterization of anthocyanins in grapes
Grapes are rich sources of anthocyanins and bioavailable flavonoids. Grapevines are one of the world’s most grown economically important fruit crops. Currently, there are more than 10000 grape cultivars deposited in germplasm collection [52, 53]. Among them, the cultivars of
2.4. Anthocyanins relevant to berry quality
During their growth, grape berries follow a double sigmoid curve [68]. Veraison is the unique stage of berry development, representing the transition from growth stage to ripening. Once the grape berries enter to veraison, many physiological and biochemical changes occur. The grape cells completely redirect their metabolism to production of secondary metabolites, necessary to prepare berries for reaching the stage of physiological maturity. During this stage, the chlorophyll in berries has been completely lost and the biosynthesis of flavonoids, including anthocyanins is promoted [68]. Microarray analysis showed remarkable overexpression of genes involved in flavonoid biosynthesis and particularly in anthocyanin production during veraison and ripening in
3. Transcriptional regulation of anthocyanin biosynthesis in grapes
Anthocyanin biosynthesis in grapes commences only when ripening of the berry begins (termed véraison) and normally continues throughout the ripening phase of growth. Anthocyanin biosynthesis pathway in grapes has been greatly investigated, including intracellular transportation and accumulation [18, 76-78]. Most of the structural genes have been isolated, cloned and characterized, and there is valuable information available on the mechanisms that regulate their expression within the plant cell [29, 44, 79]. Multiple regulatory genes under the complex regulation are responsible for the synthesis of anthocyanin at the transcriptional level [17, 18, 80]. The early biosynthetic genes (EBGs), which are upstream of the anthocyanin biosynthetic pathway, are regulated by several different families of genes called the Myb transcriptional factors, Myc transcriptional factors (encoding basic helix–loop-helix proteins, bHLH) and WD40-like proteins [40-42, 81]. On the other hand, late biosynthetic genes (LBGs), which are downstream leading to anthocyanin formation through glycosylation and subsequent modification (methylation and acylation) are under the specific control of several regulatory factors. Specific regulatory genes have been identified and characterized in
Recent studies in grapevine indicates that VvMYBA1 and VvMYBA2 transcription factors regulate UFGT gene, which plays a crucial role in the synthesis and accumulation of anthocyanins [56, 88-90], also identified another key R2R3-MYB protein that regulates proanthocyanidins (PA) synthesis in berry skin and seeds. But it is important to note that regulatory genes that control the expression of genes that encodes enzymes located upstream of UFGT have not been identified. It has been suggested that there is a contribution of at least two distinct regulatory complexes involved in the early and late steps of berry development [61]. Another study [86] also revealed that a MYB gene named
4. Muscadine grape cells as model system
Muscadines (
4.1. Genetic modification: A promising strategy to up-regulate or down-regulate the production of anthocyanins in muscadine grape cells
Genetic engineering of plants has lots of benefit in the agricultural field [95-97]. It contributes to an efficient and cost-effective way to produce a wide array of novel, value-added plant and food products in an environmentally friendly manner. Most scientists including Butelli et al. [98] have highlighted the interest of producing crop plants and their products enriched with health-promoting natural compounds. These compounds include anthocyanins and flavonoids, which have become the targets for improving the nutritional value of foods. This requires an in depth knowledge of the molecular mechanisms underlying the biosynthetic pathways of secondary metabolites in plants as demonstrated by [98]. Even though there are some plants that contain high levels of anthocyanins such as blueberries, there are some species where the accumulation of secondary metabolites is not enough. This is why genetic engineering has been used as a strategy to modify flavonoid biosynthesis in order to enhance flower pigmentation in ornamentals and fruit plants [99-103]. Genetic engineering has become increasingly important worldwide because it provides significant improvements in the quantity, quality, and acceptability of the world's food supply and may be the best source for food security [5, 104]. Currently there is an increased production of plant-based products with an enhanced antioxidant capacity, which is facilitated by this technology [103]. An example of this approach is in tomato, which is also an important vegetable crop worldwide [98, 105, 106]. Several transgenic approaches have been used to enhance the accumulation of flavonoid levels in grape berries and tomato fruit by overexpressing either the structural or regulatory genes involved in the biosynthetic pathway [87, 107]. Most of these studies have been carried out
4.2. Modification of the anthocyanin pathway using regulatory genes
The final assembly of secondary metabolites in plants is determined by the coordinate transcriptional control of structural biosynthetic genes. Based on the information provided in [112], modulation of the rate of initiation of mRNA synthesis depends on the specific transcription factors, which interact with promoter regions of targeted genes. The regulatory genes that control the pattern and intensity of anthocyanin pigmentations through regulating the expression of several flavonoid-anthocyanin structural genes have been identified in many plants [16, 113-115]. There are two families of transcription factors i.e
In addition to the over-expression of the transcription factors, the suppression or negative regulators of flavonoid biosynthesis have been described [123]. For instance, high pigmented phenotypes (hp-2) in tomato were revealed when Bino et al. [123], mutated the DE-ETIOLATED1 gene (DET1). The fruits produced from these mutants are dark, and it is because of the elevated levels of flavonoids and carotenoids. The suppression of the regulatory gene
4.3. Modification of the anthocyanin pathway using structural genes
The regulatory gene families MYB and MYC control the structural genes within the grape anthocyanin biosynthetic pathway [87, 107]. But it is important to note that, the way in which the structural genes are regulated in grape berry skins appears to be different from the patterns observed in snapdragon, petunia, and maize [125-127]. There are two ways in which the pattern of gene expression in grape berry skins could be explained in relation to regulatory genes; 1.) early biosynthetic genes, which induces the expression of all of the structural genes except UFGT, and 2.) late biosynthetic genes that results in the induction of expression of all structural genes [1]. Alternatively, two types of regulatory genes may be present, one that controls expression of PAL, CHS, CHI, F3H, DFR and LDOX and another that induces UFGT gene expression [43]. This means that the regulatory gene that controls expression of PAL, CHS, CHI, F3H, DFR, and LDOX is expressed early in berry development. But it is crucial to note that many studies have identified UFGT, as the major control point to anthocyanin biosynthesis in grape berry skins, and this control is later in the pathway than has been observed in the studies of maize, petunia, and snapdragon anthocyanin biosynthesis.
Irrespective of the function of the regulatory genes, ectopic expression of the structural genes can also enhance the accumulation of anthocyanins. A study carried out by Muir et al. [108] determined that an ectopic expression of the Petunia
4.4. Blocking specific steps in the anthocyanin biosynthetic pathway using RNAi: control of the flavonoid flux
Silencing the structural or regulatory genes on the anthocyanin pathway in muscadine requires the following steps: 1) isolation of the endogenous structural or regulatory gene; 2) construction of the transformation cassettes using structural or regulatory-gene fragments as transgenes; 3) transformation of transgenic red cells via
The flow of genetic information dictates that “DNA is transcribed into RNA that is translated into a protein” (Fig 7A). Flavonoid biosynthetic proteins are produced using this concept. To shift the metabolic flux in muscadine grape cells, one can consider either 1) over-expressing the genes on the flavonoid pathway or 2) to knock out the production of the flavonoid proteins. Blocking the production of flavonoid proteins can be done by interfering with the flow of the genetic information. For example, to eliminate the production of the muscadine DFR protein (Fig 7B), we could interfere either at the mRNA transcription level (transcriptional gene silencing [TGS]) or at the post-transcriptional level (PTGS). The advancements made in genetic engineering have led to the possibility of knocking out the production of specific proteins in organisms by downregulating and/or silencing the genes encoding these proteins. Strategies developed to downregulate genes in plants include mutation-based reverse genetics [129], gene targeting [130], antisense RNA [131], cosuppression [95, 132], and RNA interference (RNAi) [133]. Genetic and biochemical evidences suggest that antisense-mediated gene silencing, co-suppression, and RNAi are all inputs into a common RNA silencing pathway triggered by the formation of a double-stranded RNA (dsRNA). This pathway, called PTGS, is characterized by accumulation of 21 to 25 nucleotides, small-interfering RNAs (siRNAs), sequence-specific degradation of target mRNA, and methylation of target gene sequences [134]. A typical example is demonstrated by Muir et al. [108], where they used RNAi to blocked specific metabolic conversions in the endogenous tomato flavonoid biosynthesis pathway by down-regulating the expression of specific structural flavonoid genes. In another study, Schijlen [135] also used RNAi technique to inhibit tomato
5. Key concepts for enhancing anthocyanin biosynthesis in grape cell lines
Grape cell suspension cultures have been extensively studied as an model for elucidation of anthocyanin biosynthesis pathway, for performing functional genetic studies, somatic embryo development and most importantly as an alternative source of natural colorants [1, 69, 140-142]. Cultivation of plant cells in controlled conditions offers advantages of continuous supply of high quality anthocyanin pigments. However, scientists have tried for over 40 years to produce anthocyanins in different cell systems, but until now, no commercially feasible anthocyanin producing system has been developed. Switching the production of natural pigments from the traditional approach (involving implementation of numerous agricultural activities) to modern industrial biotech factories is not an easy task. But it is obvious that for the successful realization of such biotechnological advances, research on both empirical and rational levels have to be performed [143]. To succeed in such challenges, we propose to follow a simple integrated approach based on consecutive conduction of various multidisciplinary experiments, optimization and monitoring procedures (Figure 8). As we already discussed some of the rational approaches for manipulation of anthocyanin biosynthesis on genetic level, in the next few pages, the basic principles of empirical studies are highlighted and the current progress on them has been reviewed.
5.1. Cell line development and improvement
The first step for creation of biotechnology process for anthocyanins production is the development and selection of high producing cell line. To facilitate this process several important preliminary questions needs to be answered. The most important is the right choice of plant species (respectively, the appropriate cultivar) having the necessary anthocyanins profile in both quantity and quality aspects. Recently Lazar and Petolescu [144], generated cell suspension cultures of six grapevine varieties (Burgund Mare, Cabernet Sauvignon, Merlot, Oporto, Negru Tinctorial and Pinot Noir). They cultivated them in a laboratory bioreactor and demonstrated that the growth rates and biosynthetic potential for anthocyanins production were in strong dependence of cultivar used for culture initiation [144]. Currently, most of the research on grape cell suspensions have been performed with cell lines derived from two sources –
After the choice of targeted variety, the critical step is the selection of appropriate explants, which are used for callus initiation. The type and the age of explants, as well as the environmental conditions at which they are collected are critical factors for successful initiation of
However, the non-homogenous growth of grape cell suspensions could be a serious issue for large-scale cultivation of these in
5.2. Medium optimization
The optimal balance of nutrients in cultivation medium has been found to be an essential factor, determining the success on
As the grape cell suspensions exist as mixture of colored and colorless cells, the optimal cultivation medium should be developed by the way to provide a right balance between the growth rates of both cell populations [1, 142]. Finding the right nutrients balance is often a complicated task mainly because of the observation that the colorless population usually has better growth characteristics and the colored cells showed slow growth [1, 142, 150]. Dedaldechamp and Uhel [154] isolated a cell line from colorless cells of
5.3. Effect of the pH
Since the pH is an important factor for anthocyanin stability and activities of enzyme systems in plant cells, its value in the cultivation medium is critically important for the regulation of both pigments and biomass yields. Recently, the effect of pH in culture medium was investigated on callus cultures of three grapevine varieties (Coarnă neagră, Fetească neagră and Cadarcă) [164]. The authors observed that the largest amount of accumulated anthocyanins (13.5 mg/g FW) were registered in callus culture of Fetească neagră, cultivated on the medium with the pH=4.5. When cultivated on medium with pH=9.0, the anthocyanin production by the same culture was significantly decreased (up to 3.2 mg/g FW) [164]. Suzuki and colleagues [162], investigated the growth and production of anthocyanin in grape cell suspension from
5.4. Effect of temperature
Temperature has a strong effect on anthocyanin biosynthetic pathway, since some cold regulation genes are involved on it [165, 166]. Anthocyanin accumulation in berry skins of “Aki Queen” (
5.5. Light
Light is an important controlling agent in anthocyanin biosynthesis [166]. Light has been found to induce the expression of genes, responsible for activation of the promoters of the flavonoid pathway genes (
Lazar and colleagues investigated the effect of light on anthocyanin accumulation by callus cultures from six grapevine varieties (Burgund Mare, Cabernet Sauvignon, Merlot, Oporto, Negru Tinctorial and Pinot Noir) [171]. They found that the light has a stimulating effect on anthocyanin production in all calli studied, but the amount of accumulated pigments was in strong correlation with the genotype of variety used for callus initiation [171]. In grape cell suspension of
5.6. Effect of growth regulators
Availability of growth regulators (auxins and cytokinins) in cultivation medium are essential for ensuring the growth and to determine the levels of produced secondary metabolite by
5.7. Effect of elicitors and precursors
Application of different (biotic or abiotic) elicitors has been proved to be an effective strategy for enhancement of the production of secondary metabolites related to the plant defense system [175]. Anthocyanin biosynthetic pathway as a part of phenylpropanoid metabolism of plant cells could be significantly manipulated by application of different elicitors or feeding with specific precursors. Treatment of cell suspension culture of
6. Commercialization and applications of grape cell products
The increased demand of natural colorants and nutraceuticals determines the needs for development of alternative technologies for supply of such additives. The anthocyanins, produced by grape cell suspensions, represent a very attractive class of natural compounds, which could find application in food industry (as colorants), pharmacy (as nutraceuticals and therapeutic compounds) and in cosmetics (as UV protectors, antioxidant and anti cancer compounds). Biotechnological production of grape anthocyanins presents significant economical benefits. Cormier and colleagues calculated that the cost of 1 kg anthocyanins, produced by two-stage cultivation process of grape cell suspension in bioreactor with working volume of 155,000 L can cost almost the half of the price of such amount of anthocyanins, produced by the extraction of grape skins ($ 931 per 1 kg of anthocyanins from grape cell suspension, compared to $ 2,083 per 1 kg of anthocyanins, produced by grape skin extraction) [142]. However, the specific requirements of the available grape cell suspensions significantly complicate the scale up of the cultivation process, which is the serious restriction for realization of such biotechnological process.
6.1. Pharmaceutical applications
Anthocyanins have great potential for application in pharmaceutical products both as nutraceuticals and as therapeutic compounds. Frequent ingestion of anthocyanins could provide various health benefits including reduced risk of coronary heart diseases, anti-carcinogenic activity, antioxidant activity, reduced risk of stroke, anti-inflammatory effects etc. [13, 34, 184-186]. Biological activities of anthocyanin pigments have been already discussed in several excellent reviews [184, 187, 188]. Their pharmaceutical value has been additionally increased due to their high bioavailability. However, the administration and metabolism of anthocyanins
6.2. Food industry
The world market of natural food colorants expands with the annual growth rate of 4-6% [142]. In USA 4 of the 26 colorants approved by the food administration, that are exempt from certification, are based on anthocyanin pigments [34]. In European Union, all anthocyanin-containing colorants are classified as natural colorants under the classification E163 [191]. Currently most of the worldwide anthocyanins supply comes from processing of grape pomace, which is a waste product from winemaking. But in European Union other plant sources such as red cabbage, elderberry, black currant, purple carrot, sweet potato, and red radish are also allowed [192]. Anthocyanins, produced by grape cell suspensions can be a promising alternative supply of natural colorants. It has already been demonstrated that the produced pigments by the grape cell suspensions undergo significant structural modifications. Grape cell suspensions accumulates higher levels of metabolically more evolved structures (methylated and acylated anthocyanins). Acylated anthocyanins are suitable for application in food products, mainly because of the improved color stability compared to non-acylated structures [72]. Moreover, the grape cell suspensions can also produce elevated levels of beneficial phenolic compounds such as flavonoids, stilbenes, phenolics, etc., which are capable of increasing the added value of the final additive. The overall metabolite profile of grape cells in combination with the lack of microbial and toxic contaminations will give the potential for development of new types of food additives if the entire cell suspension biomass are utilized.
6.3. Cosmetic industry
The commercial interest of cosmetic companies to apply plant additives, derived by biotechnological cultivation of plant cells to their products has increased remarkably in the last few years [193]. The addition of plant cell derived extracts in cosmetic products has been considered as a powerful approach used to increase their health benefits. Several plant extracts have been added to various cosmetic products as moisturizers, antioxidants, whitening agents, colorants, sunscreens, preservatives etc. [193]. With the advancement of plant cell biotechnology, more and more cosmetic companies have been attracted for application of additives, based on plant cell suspensions. Recently the application of so-called plant “steam” cells attracts industry’s attention [193]. In the last few years, the French company “Sederma” launched the product “Resistem™” based on application of
7. Conclusion and future prospects
The approaches described in this chapter can be effective in improving novel anthocyanin-derived metabolites in grape cell suspensions. Continuous study and exploitation of the knowledge of grape cell lines and their control mechanisms will open up new possibilities for metabolic engineering of the anthocyanin biosynthesis pathway. In parallel, the recent achievements in bioengineering with plant cell suspensions and the improvements of the existed bioreactor designs discovers new prospectives for commercial realization of anthocyanin producing technology based on cultivation of grape cells. This is a research area that is growing and gaining interest in the analysis of plant-based health-related compounds. Therefore, the full impact of metabolomics on muscadine research is yet to be experienced. But this chapter serves as a starting point for scientists who are interested in cell cultures from muscadine grapes.
Acknowledgments
The authors are grateful to the Florida A&M University College of Agriculture and Food Science. The research work has been done with the financial support of USDA/NIFA/AFRI Plant Biochemistry Program Grant # 2009-03127.References
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