Root Cultures for Secondary Products

Plants are source of many high-value secondary compounds used as drugs, food additives, flavors, pigments and pesticides. The production of these compounds in nature faces to many difficulties because of the dependence on weather, soil … Furthermore, these compounds are usually limited by species, periods of growth or stress. The utilization of plant cells in vitro for the secondary compounds has gained increasing attention over past decades. However, the yield is still low, probably due to the degree of cell differentiation. Therefore, root culture is focused on research as an alternative to cell cultures to produce secondary compounds because of high rate proliferation, great potential in the production with high and stable yields. Hairy roots and adventitious roots have a high ability to biosynthesize secondary compounds in vitro with high and fairly stable in yield in comparison with plant cell suspension cultures. Nowadays, it is feasible to expand the scale of root cultures in bioreactors, which makes it possible to produce secondary compounds on an industrial scale.


Introduction
Plant secondary products are natural sources of bioactive compounds which used in traditional medicine and in industrial applications. In 1976, Farnsworth and Morris said that: higher plants-the sleeping giant of drug development [1]. Indeed, many chemicals derived from plants are important drugs, which are used as antibacterial and antitumour agents. Furthermore, they are used in antioxidant foods … Besides, natural products presented chemical structures, which are very important for scientists to pursue new chemical for drugs [2]. In plants, these valuable compounds are usually limited by species, periods of growth or stress and the yield is still low. The production faces to many difficulties because of the dependence on weather, soil …. So the utilization of plant cell, tissue and organ culture for these compounds has gained increasing attention over past decades.

Plant primary and secondary products
Plants synthetize efficiently organic compounds via photosynthesis from inorganic materials and the pathways involved are metabolic pathways. They are primary metabolism and secondary metabolism. Carbohydrates, lipids, proteins and nucleic acids are necessary for normal growth, development, and reproduction

Plant cell culture for secondary products
Plant cell culture techniques provide a reliable and predictable method for isolating valuable secondary products at high efficiency within a short time comparing to the whole plants in vivo. This provides a continuous, stable and economical production of secondary products independent of geography and climate [8].
To stabilize the raw materials for pharmaceutical industry, plant cell culture is emerging as an alternative bioproduction system. This technology offers an attractive potential to produce valuable secondary products such as ajimalicine [9], artemisinin [10], ginsenosides [11], taxol [12], resveratrol [13].
A suspension culture consists of isolated cells and cell aggregates dispersed and growing in a moving liquid medium. It used to be proved as an effective biosystem to produce valuable secondary products for commercialize. However, in most cases, for the large scale production, there are some troubles because of the instability and non-uniformity of the undifferentiated cells in liquid culture.
Adventitious root cultures show a higher constancy in the production of these compounds with more rapid growth than cell suspension cultures [14]. In addition, bioreactor system for root cultures has emerged as a technology with possible commercial applications [15]. In aseptic environment, suitable phytohormoneaugmented medium is demanded for adventitious roots formation and proliferation. In another way, hairy roots (transformed roots) derived from the infection of a plant by Agrobacterium rhizogenes -can strongly proliferation in medium without phytohormone, that is a promised biosystem for producing valuable secondary products in large scale [16].

Adventitious root cultures
Adventitious roots are roots that arises from any part of plant other than the radicles or the root axis. The formation of adventitious root needs a combination of a complicated molecular process involving numerous of endogenous and exogenous factors [17]. Adventitious roots appear in response to stress conditions, such as flooding, nutrient deprivation or wounding [18]. In vitro, the formation of adventitious roots responses to wounds and exogenous phytohormones, especially auxin (Figure 3) [19]. The induction of adventitious roots is promoted by high auxin and low cytokinin levels. There are three phase in adventitious root formation: induction, initiation and extension [20]. Auxin promotes adventitious root initiation but decreases the elongation. Root elongates when auxin concentration decreases. The application of auxins strongly increases the number of roots [21].
IBA (indol butyric acid) is most commonly used for rooting in vivo and in vitro. The other auxins used commercially are IAA (indol acetic acid) and NAA (naphthalene acetic acid) [22]. 2,4-D (2,4-Dichlorophenoxyacetic acid) is rarely used for rooting but usually used for callus initiation. The commonly cytokinins used are BAP (benzylaminopurine) and kinetin. The appropriate concentration of auxins and cytokinins in rooting depends on species, individuals and organs.
There are many scientific articles related to adventitious root cultures have been published. There are many factors that effect on rooting such as explants (type, age), exogenous phytohormones, light, organic supplements, … The process of induction and differentiation of rooting can be controlled by changes in endogenous auxin concentrations and exogenous auxins (type and concentration) [23]. The rooting of monocotyledons usually need exogenous auxins only, but dicotyledons need auxins supplemented with cytokinins. Mineral media, source of carbon, light are also important. The requirements of nutrients and exogeneous phytohormones depend on species and physiological age of explants in initiation and proliferation phase. However, the secondary products biosynthesis phase may need a different nutritional and phytohormone requirement.  Adventitious roots formed from all kinds of explants of Beta vulgaris seedlings even on free phytohormones medium. The response of root explants with auxins was better than the others. Hypocotyl explants were more suitable than cotyledon explants in adventitious root formation. The numbers of root per explant were different with the different kinds and concentrations of auxins. NAA was suitable for the initiation of roots hypocotyls and cotyledons. Whereas, IAA at various concentrations were suitable for root induction from root explants. Roots on medium with NAA were red with many root hairs, roots with IAA treatments appeared with a thicker shape and brighter red color (Figure 4). However, callus could be observed in hypocotyl and cotyledon explants and shoots formed from any treatments in hypocotyl explants [24].
The advances in plant cell, tissue and organ culture have resulted in the production of high amounts of high value secondary products [25]. Due to the rapid growth and stability in secondary metabolites production, adventitious root cultures are considered as the most promising method for biomass production [26]. Root cultures show better biosynthetic ability than plant cell suspension cultures, in a suitable phytohormone supplemented medium, with stable yield of secondary products [27]. So, adventitious roots are interested in order to increase biomass in vitro especially medicinal plants to produce bioactive compounds. Plant roots are the main raw materials of herbal drugs (about 60% of herbal medicinal plants applied for ethnomedicine needs). As a result of which, adventitious roots cultures have the potential to be developed as a strategy for large-scale bioactive compound production [28]. Establishing adventitious roots by liquid cultures would accelerate large-scale biomass and conservation in addition to supplementing pharmaceutical products [29].
Secondary products biosynthesis in vitro is effected by many factors: phytohormones, carbon sources, mineral elements, light … In liquid cultures, an important factor that effected on the growth of roots must be tested: initial inoculum density. The initial inoculum density effected on biomass and betalains accumulation of B.vulgaris L. roots in liquid culture. The inoculum density 3 g/L seemed be so low that did not sufficiently maintain betalains biosynthesis while 5 g/L and 7 g/L inoculum density almost showed more appropriate for root proliferation as well as betalains accumulation (Figure 5) [24].
The optimal condition for initiation and proliferation of adventitious roots from young Aloe vera leaves were 0.5 mg/L NAA and 0.2 mg/L BA in Murashige and Skoog (MS) medium. But aloe-emodin concentration was higher on B5 medium (133.08 ± 0.12 μg/g) than on MS medium (3.56 ± 0.26 μg/g) [30]. Andrographis paniculata adventitious roots were induced directly from leaf segments of on solid MS medium with 5.3 μM NAA but grew well and accumulated andrographolide in MS liquid medium with 2.7 μM NAA within four weeks. Fresh biomass increased seven-fold along with 3.5-fold higher andrographolide compared to natural plants [31].
Adventitious roots from Morinda citrifolia leaf explant were initiation on medium with 1.0 mg/L IBA. The highest number of roots were induced under red light, followed by blue light and lowest under far-red light. In the other hand, catalase and guaicacol peroxxidase activities were highest under red light, followed by fluorescent light and lowest under red + blue light. Moreover, superoxide dismustase activity was not influenced by light sources [32].
To enhance the production of valuable secondary products from adventitious cultures, many strategies were approached: optimization of medium and physical factors, carbon source, elicitation, precursor feeding, permeabilization and immobilization. Among them, elicitation seems to be the best solution to enhance secondary metabolites productivity in plant cell and organ cultures. Elicitor is a substance which initiates or enhances secondary biosynthesis of a living cell system when introduced in small concentration [33].
In plants, elicitor molecules attach to special receptors located on plant cell membranes. These receptors can recognize the molecular pattern of elicitors and activate intracellular defense via signal transduction pathway (Figure 6). The response results are enhancing the synthesis of metabolites which reduce damage and increase resistance to pest, disease or environmental stress [34]. Elicitors can be divided into two types abiotic and biotic according to basic nature. Abiotic elicitors include of substances that are of nonbiological origin, they are grouped in physical (thermal stress, salt tress, drought, osmotic stress) chemical (heavy metals, minerals salts, gaseous toxins) and hormonal (methyl jasmonate, salicylic acid) factors. Biotic elicitors are the biological origin substances of that comprise polysaccharides from   Many kinds of elicitor (yeast extract, methyl jasmonate, AgNO3 and sorbitol) were investigated to adventitious roots cultures of Perovskia abrotanoides Karel. Biomass and production of cryptotanshinone and tanshinone IIA were estimated. Elicitors had no significant effect on biomass (dry weight). The highest concentrations of cryptotanshinone and tanshinone IIA were achieved with 200 mg/l YE and 25 μM AgNO3, respectively. MJ moderately promoted tanshinone accumulation. Sorbitol was almost ineffective in enhancing tanshinone content. Cryptotanshinone formation was stimulated more significantly by elicitation than tanshinone IIA [38].
Root cultures of Datura stramonium were treated with copper and cadmium salts as elicitors. With the concentration at 1 mM, both Cu 2+ or Cd 2+ have been found to induce the rapid accumulation of high levels of lubimin and 3-hydroxylubimin (sesquiterpenoid). These compounds were undetectable in unelicited cultures. However, no change was seen in the alkaloid content (tropane alkaloid) of the system when treatment with Cu 2+ or Cd 2+ [39].
Adventitious roots of Glycyrrhiza uralensis were cultured MS liquid medium for the accumulation of secondary metabolites and salicylic acid has been used as an elicitor. The addition of 1 mg/L salicylic acid significantly enhanced the concentrations of glycyrrhizic acid (0.31 mg/g), glycyrrhetinic acid (0.14 mg/g) and polysaccharide (159.29 mg/g) in the adventitious roots and the contents were 2.58-fold, 1.27-fold, and 2.07-fold respectively over the control. Furthermore, the concentration of total flavonoid (9.40 mg/g) was observed with 2 mg/L salicylic, which was 2.68-fold higher than the control [40].
Aspergillus niger, Alternaria sp., Fusarium monoliforme and yeast extract were added to leaf-derived root cultures of Datura metel L., established on B5 medium with 1.2 μ M IAA, to study the influence of biotic elicitors on the growth and production of hyoscyamine and scopolamine. Besides, salicylic acid, AlCl 3 , CaCl 2 , NaCl and Na 2 SO 4 were used as abiotic elicitors. The hyoscyamine and scopolamine concentrations were 1.39 mg/g dw and 0.069 mg/g dw, respectively in control cultures. The highest hyoscyamine (4.35 mg/g dw) and scopolamine (0.28 mg/g dw) accumulation was obtained in cultures treated with 500 μM salicylic acid. 3.17 mg/g dw hyoscyamine and 0.16 mg/g dw scopolamine were observed in treatment with 0.75 g/L yeast extract and 2.49 mg/g dw hyoscyamine and 0.11 mg/g Dw scopolamine were in treatment with 250 μM AlCl 3 [41]. Many kinds of elicitors were tested in adventitious root cultures. The effects depended on species and other factors ( Table 1).
The regulation of metabolic processes in plants is highly dependent on carbon source, so plant cells and tissue are quite sensitive to sugar concentration in nutrient medium [54]. In vitro plant cells are heterotroph, although in many cases they canlive as mixotroph thanks to artificial lighting and chloroplasts. Therefore, the supplement of sugar is necessary. Saccharose is the most common sugar, which accelerates the growth of biomass, which is commonly used in the concentrations of 2 to 5%, but also depends on the purpose of culture [55].
In broccoli (Brassica olearacea var. capitata) adventitious root cultures, the proliferation of roots enhanced with the increasing of saccharose from 20 to 40 g/L and decreased with saccharose 50 g/L. The color of roots was white with saccharose 20 and 30 g/L and pale yellow with saccharose 40 and 50 g/L (Figure 7) [56].
The role of saccharose can be explained by the effect on tubulin, one kind of protein presents throughout the growth and development of the cell. Tubulin controls the cell shape, cell division and intracellular transport via genes tual and

Pectins
Anthraquinones [48] Gynura procumbens (L. incw1. These genes are only exhibited with the presence of saccharose [57]. When the concentration of saccharose in medium is too high, it's difficult for cell to absorb nutrients so the proliferation will decrease. Beside the role in biomass proliferation, carbon source also effects on secondary products biosynthesis. According to Miao et al., glucose is also an inducer of glucosinolate biosynthesis. Glucosinolate biosynthesis is mediated indirectly by XK1 (hexokinase 1) and/or RGS1 (G1 protein regulatory signal) through MYB28 and MYB29 translation factors, both of them are induced by glucose. As a signaling molecule, glucose can regulate growth, development, metabolism and resistance to environmental stress of cells [58]. Glucose is released from the saccharose during autoclaving as well as by invertase which takes part to glucosinolate biosynthesis [59].

Hairy roots
Hairy roots derived from the infection of plant by Agrobacterium rhizogenes, a Gram-negative soil bacterium. Hairy roots can be obtained from a wide variety of plants and be well interested because of the ability of valuable secondary metabolites production. Hairy roots can produce and secrete complex active glycoproteins and organic compounds from a wide variety of plants. Nowaday, hairy roots have positioned as effective biological systems in pharmaceutical industry due to the development of fully controlled large-scale bioreactors [60].
Agrobacterium sp. are agents of disease in plants. Agrobacterium tumefaciens cause crown gall disease and Agrobacterium rhizogenes cause abnormal roots (rootmat disease) in dicotyledonous plants. Hairy roots induced by Agrobacterium rhizogenes are very similar to wild-type roots in structure (Figure 8) except some characteristics: lateral branching, root hairs are longer, more numerous,  have an agravitrpic phenotype and genetic stability (Figure 9). In especially, the ability of hairy roots is growing quickly in vitro in the absence of exogeneous phytohormones.
The mechanisms for crown gall or hairy root formation are very similar, depend on Ti-plasmid (tumor inducing plasmid) and Ri-plasmid (root inducing plasmid) respectively. In Agrobacterium, a portion of Ti-plasmid or Ri-plasmid, T-DNA (region bounded by 25 bp direct oligonucleotide repeats-right border and left border) is transferred to the plant cell, randomly integrated into the host genome and expressed. Vir genes are very important to the infection of this bacterium to the plant cell (Figure 11).
There are two kinds of Ri-plasmid: agropine and mannopine based on the compounds that are synthesized by the transgenic plant tissue [64]. Agrobacterium recognizes some signal molecules (phenolic compounds) excreted by the wound in plant and attached to it. In the Agropine, Ri-plasmids consist of two copies: left T-DNA (TL-DNA) and right T-DNA (TR-DNA), each copy is transferred independently (Figure 12). Encoding genes in T-DNA are bacterial origin but they can express in infected plant cells because of eukaryotic regulatory. Genes of auxins synthesis are ascribed to the TR-DNA. The right T-DNA of Ri-plasmid contains two   tms1 and tms2 (aux1 and aux2). The TR-DNA also contains genes for agropine synthesis (ags). The TL-DNA has been sequenced and a total of 18 open reading frames (ORF1-ORF18) have been identified   [65]. For the formation of the hairy roots, four rol genes (rolA, rolB, rolC, and rolD) are very important. These genes correspond to reading frames ORF10, ORF11, ORF12 and ORF15 [66]. The products of rol genes have specific functions in the hairy roots formation; among them, rolB gene seems to be the most relevant in the induction. Also the rol-genes have a big influence on the phenotype of hairy roots [67]. RolA protein is suggested as a transcription factor that has been proposed to participate in the metabolism of gibberellins. The rolA gene is also reported to be responsible for changes in polyamine metabolisms. The rolB gene is important in the mechanism of adventitious root formation in plants. The adventitious roots induced by the rolB gene produce lateral roots in cell plant cultures, that indicates that the rolB protein has an effect on the formation of both lateral and adventitious roots. The rolC gene effects on the plant size and architecture, these include height decreasing, internode elongation, male fertility, apical dominance and increasing number of flowers. Other effects are the changes in leaf size, color and shape that increasing their ornamental value. The RolD is suggested to exert a positive effect on flowering by inducing a striking earliness in the flowering process and increasing the number of flowers [68].
Hairy roots grew more rapidly and produce higher levels of secondary products than the adventitious root obtained by hormonal control. One of the final goals of hairy root cultures is to produce valuable plant secondary products in large-scale bioreactors [69].
Hairy roots have different shapes depends on the Agrobacteroum rhizogenes strain that infected. Hairy roots were established by the infection of six different Agrobacterium rhizogenes strains to two varieties of Catharanthus roseus. Fourty seven hairy root clones were recorded. Growth rate and morphological appearance of hairy roots were wide showed (Figure 13) [70].
Hairy roots from root discs of Panax ginseng C.A. Meyer were obtained after the infection of Agrobacterium rhizogenes A4. Hairy roots displayed three phenotypes (three lines): the first lines showed the characteristic traits of hairy roots (HR-M), the second were callus-like (C-M) and the third were thin, without branching (T-M) (Figure 14). HR-M and C-M root phenotypes presented the highest biomass. The highest ginsenoside production was achieved by HR-M root lines, followed by C-M and the lowest yield was found from T-M root phenotype [71].
Hairy roots were induced from Rhaponticum carthamoides leaf explants by the transformation of Agrobacterium rhizogenes strains A4 and ATCC 15834. A4 strain was more appropriate than ATCC 15834 in the formation of transformed roots. Hairy roots systems were established in liquid media (WPM, B5, SH) with full and half-strength concentrations of macro-and micronutrients. Two different lighting conditions (light or dark) were tested on the biomass of hairy root line (RC3). The highest biomass was obtained in WPM medium under periodic light. The content of caffeoylquinic acid and their derivatives was raised in hairy roots grown in the light. Besides, the biosynthesis of flavonoid glycosides such as quercetagetin, quercetin, luteolin, and patuletin hexosides was detectedin the light. Chlorogenic acid, 3,5-di-O-caffeoylquinic acid and tricaffeoylquinic acid derivative were found as the major compounds present in the transformed roots [72].
The efficiency of transformation depends on many factors: type and age of explant, the strain, density and growth stage of Agrobacterium rhizogenes, acetosyringone concentration, the pre-culture time, the infection time... Plant secondary production by hairy roots process: 1. Hairy roots induction and proliferation.
2. Hairy roots in liquid phase: nutrient medium optimization, several strategies can be used to improve the yields of target compounds.
3. Bioreactor stage: batch / fedbatch or continuous culture. Optimization airflow rate, temperature, pH…. To improve the yield of valuable secondary products in hairy root cultures, elicitation seems to be the most effective strategy. Hairy root cultures are preferred for the application of elicitation because of their stable genetics and biosynthesis and high growth rate in non-phytohormone medium. Elicitors act as signals that were recognized by elicitor-specific receptors on the plant cell membrane and stimulate defense responses during elicitation. The results are the increasing of synthesis and accumulation of secondary metabolites. The effects of elicitation depend on elicitor type, concentration, duration of exposure and treatment schedule ( Table 2).
Panax ginseng C.A. Meyer hairy roots from roots, stems, and leaves induced by the infection of Agrobacterium rhizogenes (KCTC 2703) were propagated in 5-liter cone type bubble bioreactors containing MS media supplemented with 2.0 mg/L NAA and 30 mg/L sucrose. Jasmonic acid in various concentrations was added to the culture system after 30 days of culture to increase ginsenoside concentration. Total ginsenoside concentration increased with the increasing of jasmonic acid concentration, but the root growth was inhibited with high concentration. Total productivity was greatest at 2.0 mg/L jasmonic acid but there was the difference in groups of ginsenoside. Ginsenosides in the Rb group mainly increased, while those in the Rg group did not. High concentrations (5 and 10 mg/L) of jasmonic acid decreased Rg1 content but significantly increased the Rb1. In the Rb group, the Rb1 content increased more than Rb2, Rc, and Rd. [88].

Species
Elicitors Secondary products
Ag + Silymarin [75] Plumbago indica Jasmonic acid Plumbagin [76] Glycyrrhiza Panax quinquefolium Yeast extract Ginsenosides [85] Ocimum tenuiflorum L Yeast extract, Methyl jasmonate, Salicylic acid Ursolic acid and eugenol [86] Scutellaria bornmuelleri Methyl jasmonate + chitosan Chrysin, wogonin and baicalein [87]  In another experiment, peptone and jasmonic acid were used as elicitors to promote ginsenosides accumulation in Panax ginseng C.A. Meyer hairy roots induced by the infection of Agrobacterium rhizogenes (KCTC 2703) to root explants. Root system was cultured in phytohormone-free Murashige and Skoog liquid medium. Jasmonic acid in the range 1.0-5.0 mg/L strongly improved total ginsenoside production. Peptone (300 mg/L) showed good effects on ginsenoside concentration but weaker than that of jasmonic acid. The Rb group of ginsenoside content was increased remarkably by jasmonic acid, while Rg group ginsenoside content changed slightly compared to controls. However, jasmonic acid also strongly inhibited hairy root growth [89].
Node explants of Vitis vinifera subsp. sylvestris were used as materials for the hairy root induction by Agrobacterium rhizogenes ATCC 15834. Hairy roots were immerged in ½ B5 medium without phytohormone. Methyl jasmonate and other elicitors were used to enhance resveratrol biosynthesis of hairy roots. The result showed that the resveratrol production of hairy roots was higher than natural roots. Especially, the production of resveratrol increased with the present of elicitors. There was a significant difference in inducing resveratrol production between the elicitors. The treatment with 3 mM acetic acid led to the highest resveratrol content and methyl jasmonate seemed to be less effective than the others [90].

Conclusion
Adventitious roots and hairy roots are promising materials for the production of valuable secondary compounds of plants which are used in pharmaceutical, food and cosmetic industry. The chemical characteristics of these compounds are the same as that in natural plants but the yields are proved higher. Furthermore, there are many investigations which focused on improving bioreactor for root cultures to raise their quality and productivity.