The application of elicitors on secondary products of adventitious root cultures.
Abstract
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.
Keywords
- adventitious roots
- Agrobacterium rhizogenes
- elicitors
- hairy roots
- secondary products
1. 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.
2. 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 of plants (primary products). Besides, there is a large, diverse array of organic compounds that have no direct function in growth and development of plants. These substances are known as secondary products (secondary compounds, secondary metabolites or natural products) [3].
Secondary products are restricted distribution in the plant kingdom, that is found in only one plant species or related group of species. For many years, these compounds were thought to be simply functionless end products of metabolism or metabolic wastes. But now, secondary products have been suggested to have important ecological functions in plants. They protect plants against being eaten by herbivores and against being infected by microbial pathogens (Figure 1). Furthermore, they serve as attractants for pollinators, seed dispersing animals and as agents in the competition of plants [4].
Secondary metabolism is connected to primary metabolism by using intermediate products and biosynthetic enzymes derived from primary metabolism. Secondary compounds are synthesized through mevalonate, non-mevalonate (MEP (methylerythritol phosphate) shikimate and malonate pathway (Figure 2). These metabolisms rely on environmental conditions, physiological states and stages of plant growth, and yields are often very low.
There are many ways of classification of secondary products, but in general, they are divided into three chemically distinct groups: terpenes, phenolics, and nitrogen containing compounds.
The terpenes (terpenoids, isoprenoids) seem to be the largest class of secondary products. They are biosynthesized from acetyl-CoA – intermediates of many biological reactions. Terpens are widely used in pharmaceuticals, food and cosmetics industries. They possess antitumor, anti-inflammatory, antibacterial, antiviral, antimalarial effects, promote transdermal absorption, prevent and treat cardiovascular diseases, and have hypoglycemic activities [5].
The phenolics in plants are a chemically heterogeneous group of nearly 10,000 individual compounds. Many kinds of phenolics are used as agents of anti-aging, anti-inflammatory, antioxidant and anti-proliferative activities. They are used as therapy agents for chronic diseases, diabetes, cancers, cardiovascular diseases … through the management of oxidative stress [6].
Alkaloids are organic compounds that contain at least one nitrogen atom at any position in the molecule, which does not include nitrogen in an amide or peptide bond. Alkaloids have a wide range of biological activities such as antiviral, antibacterial, anti-inflammatory, antitumor …. [7]. Many of these compounds possess potent pharmacological effects, for example, the well-known plant alkaloids include the narcotic analgesics (morphine, codeine, apomorphine (a derivative of morphine) used in Parkinson’s disease, the muscle relaxant papaverine, the antimicrobial agents sanguinarine and berberine. Also several potent anti-cancer drugs have been developed from plant compounds such as vinblastine, vincristine, taxol, camptothecin, colchicine … .
3. 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
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 phytohormone-augmented medium is demanded for adventitious roots formation and proliferation. In another way, hairy roots (transformed roots) derived from the infection of a plant by
3.1 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].
IBA (indol butyric acid) is most commonly used for rooting
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
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
Secondary products biosynthesis
The optimal condition for initiation and proliferation of adventitious roots from young
Adventitious roots from
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 plant cell walls (e.g. chitin, pectin, and cellulose), yeast extracts, fungal or bacterial extracts, microorganisms or saliva of insects or herbivores [35]. Methyl jasmonate is a potent elicitor in plant cell, tissue and organ culture for secondary compounds [36].
The effects of elicitors on secondary productivities depend on:
Elicitor concentration
Duration of elicitor influencing
Cell lines
Time course of elicitation
Growth stage of culture system
Phytohormone
Nutrient composition [37].
Many kinds of elicitor (yeast extract, methyl jasmonate, AgNO3 and sorbitol) were investigated to adventitious roots cultures of
Root cultures of
Adventitious roots of
Species | Elicitors | Secondary products | References |
---|---|---|---|
Cu2+, Cd2+ | Lubimin, 3-hydroxylubimin | [39] | |
Cellulase | Capsidiol | [42] | |
Salicylic acid, yeast extract, NaCl | Hyoscyamine and scopolamine | [41] | |
Methyl jasmonate, salicylic acid, chitosan | Valtrate | [43] | |
Chitosan | Anthraquinone, phenolics and flavonoids | [44] | |
Salicylic acid | Aloe emodin and chrysophanol | [30] | |
Casein hydrolysate | Ginsenoside | [45] | |
Yeast extract, AgNO3 | Cryptotanshinone, tanshinone IIA | [38] | |
Methyl jasmonate | Psoralen | [46] | |
Salicylic acid | Glycyrrhizic acid glycyrrhetinic acid polysaccharide | [40] | |
Protein fragment of more than 10 kDa | Flavonoids, glycyrrhizic acid, glycyrrhetinic acid and polysaccharide | [47] | |
Pectins | Anthraquinones | [48] | |
Yeast extract, CuSO4 1 mg/L | Quercetin, kaempferol | [49] | |
Methyl jasmonate | Saponin | [50] | |
Methyl jasmonate | Saponin | [51] | |
Salicylic acid | L-Dopa | [52] | |
Uv-B 4°C | Hypericin | [53] |
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 broccoli (
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
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].
3.2 Hairy roots
Hairy roots derived from the infection of plant by
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
There are two kinds of Ri-plasmid: agropine and mannopine based on the compounds that are synthesized by the transgenic plant tissue [64].
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
Hairy roots from root discs of
Hairy roots were induced from
Hairy roots from petiols of
The efficiency of transformation depends on many factors: type and age of explant, the strain, density and growth stage of
Plant secondary production by hairy roots process:
Hairy roots induction and proliferation.
Hairy roots in liquid phase: nutrient medium optimization, several strategies can be used to improve the yields of target compounds.
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).
Species | Elicitors | Secondary products | References |
---|---|---|---|
Jasmonic acid, Salicylic acid | Azadirachtin | [74] | |
Ag+ | Silymarin | [75] | |
Jasmonic acid | Plumbagin | [76] | |
Chitosa Methyl jasmonate, Yeast extract | Glycyrrhizin | [77] | |
Methyl jasmonate, fungal elicitors ( | Artemisinin | [78] | |
CaCl2 | Valerenic acid | [79] | |
Salicylic acid | Tanshinone | [80] | |
Methyl jasmonate | Isoflavonoid | [81] | |
NaCl, cellulase from | Ajmaline, solasodine and α-solanine | [82] | |
Daidzin | [83] | ||
Scopolamine | [84] | ||
Yeast extract | Ginsenosides | [85] | |
Yeast extract, Methyl jasmonate, Salicylic acid | Ursolic acid and eugenol | [86] | |
Methyl jasmonate + chitosan | Chrysin, wogonin and baicalein | [87] |
In another experiment, peptone and jasmonic acid were used as elicitors to promote ginsenosides accumulation in
Node explants of
4. 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.
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