Anthocyanins are a type of natural, edible pigments with strong biological activities such as anti-oxidation, anti-cancer and anti-aging. As the masses pay more attention on human health issues, their demand for anthocyanin products keeps growing. However, owing to the shortage of raw material and the instability of source supply, there are few anthocyanin products on the market and their price is expensive and it is difficult to satisfy the anthocyanin demand of consumers. Therefore, we hope to develop an in vitro biosystem that could produce anthocyanin in larger quantity and shorter terms with edible carrot cells as chassis cell. Though this expandable system, the mass production of anthocyanins could be realized.
Anthocyanins belong to a parent class of molecules called flavonoids synthesized via the phenylpropanoid pathway. They occur in all tissues of higher plants, including leaves, stems, roots, flowers, and fruits (Andersen et Jordheim, 2010). As a type of widely-distributed polyphenolic pigments, anthocyanins are water-soluble, colourful molecules of the flavonoid class. Anthocyanins’ molecular structure change with different pH and make plants exhibit a variety of colours attracting pollinators to help progeny.
Besides, anthocyanins have strong biological activities such as anti-oxidation and anti-aging, and are widely used in the field of nutrition and health care.
One of the functions of anthocyanin is to eliminate free radicals, mainly oxygen radicals, to achieve the anti-oxidant effect. Because free radicals are very active, oxygen radicals in the body will actively react with lipids in human cells to cause cross-linked polymerization of proteins and other macromolecules. Lipofuscin is one of the examples, it tends to accumulate in the skin causing senile plaques due to its insolubility in water. Similarly, the solubility of collagen in water becomes worse after cross-linked polymerization, resulting in wrinkles due to loss of skin tone, increased likelihood of rupture due to loss of vascular elasticity, weakened regenerative capacity, and blurred retina due to lipid peroxidation of the lens, inducing age-related visual impairment (blurred eyes, cataracts, etc.), all of which are symptoms of aging of the body.
Raw materials and techniques used in the anthocyanin extraction and manufacturing of goods containing anthocyanins are crucially important economically.
At present, the industrial production of natural anthocyanins is mainly based on anthocyanin-rich berries of different plants, such as grapes, Blueberry, Blackberry, cherry, Black Currant, etc. Not only do these plants have a long harvest period, they also suffer from climate change and seasonal changes. Therefore, the yield of anthocyanins from these plants is often unstable due to the instability of raw material sources. In addition, since the cultivation and growth of these plants require a large amount of farmland, the production scale is also limited in countries or regions with a large population but insufficient arable land
Currently, most anthocyanins are extracted from plants by physical or chemical means. In recent years, some new extraction methods have been developed using eutectic solvents, aquatic solvent combinations, and other chemically reactive extractions, such as extractions based on low acid concentrations.
Since the main constituent ingredient of these plant materials are other substances, such as lignin and saccharide, a large amount of production waste is caused during the production process, which not only causes economic waste, but also causes environmental pollution.
Due to their immense importance as dietary neutraceuticals, enhanced production of these anthocyanins from cell/tissue cultures have been extensively explored since the last 2 decades, (Biswas et Mathu, 2017). In all higher plants studied to date, the anthocyanin pigment pathway is regulated by a suite of transcription factors that include MYM, bHLH and WD-repeat proteins (Gonzalez et al., 2008). The MBW (MYB–bHLH–WDR) protein complexes participate in different types of controls ranging from fine-tuned transcriptional regulation by environmental factors to the initiation of the flavonoid biosynthesis pathway by positive regulatory feedback (Xu et al., 2015). Ectopic expression of a MYB or a MYB plus a bHLH gene significantly increased anthocyanin content in many plants, which has already become a technology to produce biofortified foods with enhanced anthocyanins (Butelli et al., 2008)
The maize (Zea mays) C1 gene, which is responsible for the regulation of anthocyanin pigmentation (Paz-Ares et al., 1987), was the first plant MYB gene cloned (Feller et al., 2011). Of plant bHLH proteins whose function has been established, many regulate anthocyanin biosynthesis (DEL, JAF13, B-peru and R-1c) (Heisler et al., 2001).
S. baicalensis, called “huangqin” in Chianise, is an important Chinese medicinal plant, it accumulates anthocyanins in its flowers, the co-pigmentation of the anthocyanins with flavones accounts for the purple-blue colour of S. baicalensis flower. (Oszmiański et al., 2004). Yuan et al. (2015) had screened RNA-sequencing databases and found that several MYB genes may be responsible for regulation of production of its flavonoids.
Agrobacterium are a type of Gram-negative bacteria commonly found in the soil. There are two often used species: A. tumefaciens and A. rhizogenes. A. tumefaciens tends to infect the injured parts of most dicotyledons or gymnosperms under natural conditions, inducing the production of crown gall tumours, while A. rhizogenes induces the production of hairy roots, characterized by the proliferation of highly branched root systems. This is why Agrobacterium is a natural plant genetic transformation system, capable of transferring and integrating exogenous genes into plant cells by inserting the target gene into a modified T-DNA region, and obtaining transgenic plants through cell and tissue culture techniques. This is Agrobacterium-Mediated Genetic Transformation (AGT). This method is now commonly used in the laboratory not only for the infection of dicotyledonous plants, but also successfully for the transformation of non-Agrobacterium host organisms such as fungi, monocots, gymnosperms and even animal cells. It is now commonly used, especially for transgenic engineering research in plants (Maokangbio et al., 2018).
Anthocyanin is a natural, non-toxic type of pigment that can be used as a food colourings. Owing to its biological activity of anti-oxidation and anti-free radicals, it has become a health care product with function of anti-aging. Thus, the social demand for anthocyanins increases.
However, due to the unstable source of raw plant materials, the occupation of limited farmland and the great quantity of production waste that coursing environmental pollution and waste of resources, the scale of the current anthocyanin industry is serious limited. The yields for this industry are far from meeting consumer demand.
Based on previous studies on the synthesis pathways and regulatory factors of anthocyanins in plants, we can completely solve these problems with in vitro culture of specific cells, tissues or organs of plants at high density indoors. Meanwhile, methods of transgenic and biosynthetic technologies can be used to regulate and promote the rapid synthesis of anthocyanins in plant cells.
Based on previously reported S. baicalensis genome (Zhao et al., 2009), we plant to isolate the MYB and bHLH regulators for anthocyanin biosynthesis, and integrate these key genes into cells of carrot, one of the most widely eaten vegetable, through the transformation mediated by Agrobacterium rhizogenes. Transgenic hairy roots, rather than whole plants with large quantity of waste, will be induced to emerged and cultured in vitro to produce anthocyanins. A high-yield and scalable anthocyanin biosynthesis cell system will be realized.
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