Abstract:
Biotechnology methods particularly tissue culture techniques offer new approaches for the in vitro production of valuable plant nutraceuticals. The large-scale production of secondary metabolites in a year-round system without seasonal constraints is one of the advantages of plant tissue culture techniques. Furthermore, enhancement of in vitro nutraceuticals production can be realized by a selection of high-producing cell lines and medium optimization. In this respect, several techniques have been adopted to improve the production of plant-derived nutraceuticals such as mutation, elicitation, precursor feeding, genetic transformation and metabolic engineering. In vitro mutagenesis of cultured cells and tissues by irradiation or chemical mutagens represents a feasible method for the improvement of nutraceuticals production. Likewise, transformation is another strategy that is used to enhance nutraceuticals in plant cells or organ tissues. At this point, hairy root culture induced by Agrobacterium rhizogenes is the most promising transformation technique used for in vitro production of valuable plant compounds. The benefits of hairy roots are high metabolite productivity, high growth rates and inherent genetic stability. On the other hand, due to its effectiveness and practical feasibility, elicitation is considered the most applied strategy for enhancing the production of desired compounds in plant biotechnology. Biotic (biological origin) and abiotic (non-biological origin) elicitors are used to increase the production of plant metabolites by different effects on the cellular processes in the plant system. Furthermore, bioreactors developed for in vitro industrial-scale production of desired compounds can be used for continuous and scaling-up plant nutraceuticals production. This article discusses different aspects of biotechnology used for the selection and enhancement production of nutraceuticals from plant cultures grown cells, in vitro.
INTRODUCTION
Nutraceutical is referred to as nutrient compounds that have medicinal properties. Nutraceuticals include antioxidants, vitamins, amino acids and dietary substances1,2. Nutraceuticals can be concentrated in the form of pills, tablets, liquids, or powders for direct consumption or use as ingredients in functional foods. Bioactive compounds which provide health benefits are also referred to as nutraceuticals, a term that reflects their existence in the human diet and the biological activity3. The term nutraceuticals, itself is a wide class that includes many categories and subcategories under it. In this respect, nutraceuticals can be classified on their natural sources, pharmacological conditions, as well as the chemical constitution of the products.
The contribution of nutraceuticals to public health is considered one of the most concerned approaches in the healthcare system. Due to their supposed safety and possible therapeutic effects, nutraceuticals have attracted considerable interest over the years. Recently, they have been explored as sustainable alternatives for the fight and reducing a large number of diseases. In this respect, nutraceuticals are used to prevent chronic diseases and postpone the ageing process. Moreover, nutraceuticals are beneficial in fighting life-threatening diseases as well as different infections4. Furthermore, research has indicated the involvement of nutraceuticals in the treatment of insomnia, digestion problems, cancer, blood pressure abnormalities, cold and cough, depression and coronary heart disease5. Otherwise, some plant-derived bioactive and therapeutics play a preventive role against the incidence of certain diseases such as cancer, ageing, inflammation and cardiovascular disorders6-8. Continuing demand from the consumer for beneficial foods leads to the exploration and recognition of new and emerging sources of nutraceuticals and functional foods. At this point, higher plants are considered a biochemical factory to produce secondary metabolites used as nutraceuticals such as flavonoids, polyphenols, alkaloids, terpenes and glycosides.
Difficulties in cultivation practices, seasonal variation in productivity and economic cost are the major hinders to large scale-production of phytochemicals from field-grown plants. To search for alternatives, biotechnological methods especially in vitro culture technique represent a potential renewable source of such compounds. Plant tissue cultures are established from different explants for the accumulation and gathering of secondary compounds such as nutraceuticals. The advantages of tissue culture technology are: (1) Desired compounds can be obtained independent of cultivation season, (2) Cultured cells will be free of contaminations (3) Automated control of cell growth of metabolite processes would reduce labour costs, (4) Isolation of the phytochemical can be rapid and efficient when compared with extraction from whole plants and (5) Biosynthetic of these phytochemicals can be in vitro regulated to maximize yields. In this respect, various biotechnological strategies such as in vitro mutagenesis, selection of high yielding lines, optimization of culture media composition and physical parameters, precursor feeding, elicitation, large scale cultivation in a bioreactor system, hairy root culture, metabolic engineering, plant cell immobilization, biotransformation, etc., have been studied for their effectiveness towards enhancement of phytochemicals production utilizing in vitro of culture different plant varieties9.
Classification of nutraceuticals: Nutraceuticals are the compounds that are found in foods, dietary supplements and herbal products and have health benefits and medicinal properties. Based on their source, chemical composition and nutrients, nutraceuticals are grouped into several classes. Also, they can be classified as traditional and nontraditional nutraceuticals. Traditional nutraceuticals are the components that deliver benefits beyond the basic nutrition of natural foods. While non-traditional nutraceuticals are the fortified components prepared by adding new characteristics for the well-being of humans. Recombinant nutraceuticals are food with added nutrients and others that are produced with the help of biotechnology. Otherwise, nutraceuticals are classified into seven different groups based on the source Table 1.
Phytochemicals as nutraceuticals: Phytochemicals have been given more attention as human nutrition because of their pharmaceutical effects. There are more than 4,000 phytochemicals catalogued and are mostly classified based on their biological activity, physical characteristics and chemical characteristics20,21. Three major classes of plant secondary metabolites are involved in these phytochemicals alkaloids, terpenes and phenolics. Moreover, there are other many phytochemicals classes recognized with health benefits such as phytosterols, thiosulfonates etc.
Terpenoids (terpenes): Plant terpenoids, the large class of natural organic chemicals are used for their aromatic qualities and play a role in traditional herbal remedies. Terpenoids contribute to the scent of eucalyptus, the flavours of cinnamon, cloves and ginger, the yellow colour in sunflowers and the red colour in tomatoes. Meanwhile, terpenoids are important for plants due to their ability to fix carbon through photosynthetic reactions using photosensitizing pigments like chlorophyll and carotene.
| Table 1: | Source and health benefits of the main nutraceuticals classes | |
| Nutraceuticals | Sources | Health benefits |
| Dietary fibre | Dietary fibre is a plant origin substance that includes polysaccharides, oligosaccharides, lignin and associated plant substances. Fibre is found in grains, pulses, fruits and vegetables | Functionally, fibres promote beneficial physiological effects including laxation and/or blood cholesterol attenuation and/or blood glucose attenuation10. The research results proved that people who eat fibre rich diets tend to have a reduced risk of certain cancers, coronary heart disease and obesity11 |
| Probiotics | The foods that contain probiotics are curd, beverages, yoghurt, cheese and pickles. Lactobacillus is the most probiotic found in yoghurt and other fermented foods | Probiotics are effective in diarrhoeal diseases, irritable bowel syndrome, stomach infections and colic diseases12 |
| Prebiotics | Lactulose, lactitol oligofructose, inulin, fructooligosaccharides and galacto-oligosaccharides are prebiotics | Prebiotics supports the probiotic growth of gut bacteria and potentially enhance digestion and metabolism13 |
| Polyunsaturated fatty acids | Polyunsaturated fatty acids contain omega-3 (essential fatty acids) and omega-6 fatty acids. Fish oils, nuts, salmon, tuna, groundnuts, oysters, flaxseeds are rich sources of essential fatty acids. However, omega 6-fatty acid is found in sunflower, soybean and corn | Polyunsaturated fatty acids are important for nerve function, blood clotting, brain health and muscle strength. Moreover, polyunsaturated fats can help reduce cholesterol levels and the risk of heart disease and stroke14 |
| Antioxidants | Vitamin C (ascorbic acid), vitamin E (tocopherols and tocotrienols) and carotenoids are considered antioxidant vitamins. Also, red wine, tea (green, black), tea (black beverage) and cocoa are other antioxidant polyphenols | Antioxidant vitamins prevent oxidation of cellular organelles, membranes, biochemical pathways leading to several degenerative diseases including cancer, cardiovascular diseases and cataracts15. Also, phenolic antioxidants decrease oxidative cell injuries and inflammatory reactions improving the brain’s health16 |
| Polyphenols | Flavonoids polyphenols are widely found in fruits, vegetables, wines, teas and cocoa as glycosides, dimers and polymers. Non-flavonoid phenolic compounds i.e., resveratrol, caffeic acid and curcumin are found in coffee beans, potatoes, apples and olive oil18 | Polyphenols have been reported to have antioxidative, anti-inflammatory, anti-mutagenic and anti-carcinogenic activities17 |
| Spices | Among the traditional spices, there is a growing interest in wasabi, horseradish, turmeric and ginger whose antioxidant properties | Spices have antioxidative, chemopreventive, anti-mutagenic, anti-inflammatory, immune-modulatory effects for the benefit of human health19 |
They are also well recognized for their role in stress response or defence mechanisms22. On the other hand, terpenoids have a wide range of biological functions and have been used in the preparation of functional foods, flavourings, bio-colourants pharmaceuticals, cosmetics, disinfectants and agrichemicals. From a healthy point of view, several terpenoids have been studied and used as pharmacological agents to benefit human health. In this regard, certain diseases such as chronic damage and growth dysregulation were protected using terpenoids compounds23. Tocopherol, α-d-tocopherol (vitamin E) has been extensively reviewed for its antioxidant potential and is also reported effective apoptotic inducer for human breast cancer cells24. Carotenoids, another important class of terpenoids are coloured (yellow, orange and red-pigmented) compounds and are present in various fruits and vegetables with a very rich antioxidant profile. More than 600 carotenoids have been found in plants and they comprise two types of molecules, carotenes and xanthophylls. Carotenoids have a tissue-specific biological activity and have been reported to protect against uterine, prostate, breast, colorectal and lung cancers25.
Polyphenolics: Polyphenols are natural compounds synthesized by plants with chemical features related to phenolic substances. Phenolic compounds can be divided into flavonoids, flavonols (quercetin), flavones (apigenin), flavanols (catechin and its derivatives, proanthocyanidins), anthocyanidins (anthocyanins), flavanones (hesperidin), isoflavones (genistein, daidzein) and non-flavonoids, stilbenes (resveratrol), phenolic acids (gallic acid, hydroxybenzoic acid, hydroxycinnamic acid, chlorogenic acid, caffeic acid, ferulic acid). There are approximately 8,000 different sub-classes of polyphenols. The most important are flavonols, flavones, flavan-3-ols, flavanones and anthocyanin18. The main sources of polyphenols are fruits, vegetables, black tea, green tea, coffee, chocolate, red wine, olives and extra virgin olive oil. Nuts, spices, herbs and algae are also potentially significant for supplying certain polyphenols26. Recognition of the antioxidant activities of many polyphenols has established a correlation with the health benefits of such compounds27. In this respect, flavonoids, the major active phenolic compounds have long been recognized to possess anti-inflammatory, antiallergic, hepatoprotective, antithrombotic, antiviral and anticarcinogenic activities. The main representatives of flavonoids polyphenols are quercetin, catechin and kaempferol. Generally, phenolic compounds have beneficial effects on several diseases including cancer, cardiovascular disease, myocardial damage and neurodegenerative disorders28. These compounds can defend the body against cancer by protecting DNA from radical damage and avert cardiovascular disease by preventing the oxidation of lipids and cholesterol29.
Alkaloids: Alkaloids are a class of naturally occurring organic compounds that contain at least one nitrogen atom. It was found that 20% of plant species contain alkaloids and the major source of the alkaloid is the flowering of the plants. Alkaloids are medicinally known as local anaesthetic and stimulants, psychedelics, anticancer drugs, cholinomimetic, spasmolysis agents, vasodilators, antiarrhythmics, antiasthma therapeutics, etc30. Alkaloids are, being present in several economically relevant plant families. Among alkaloids, glucosinolates contain sulfur and nitrogen and are derived from glucose and amino acid. Glucosinolates present in cruciferous vegetables such as broccoli, cauliflower and cabbage are widely consumed and their beneficial health-promoting effects are well established23. The current interest in glucosinolates is focused on their ability to protect against cancer since studies have shown an inverse relationship between the consumption of vegetables containing glucosinolates and the risk of cancer31. Otherwise, purine alkaloids, including caffeine (coffee), theophylline (antiasthma drug), theobromine (chocolate) and other methyl-xanthines, play a significant role in pharmacology and food chemistry32. The main effects of moderate consumption of caffeine by humans are an increase in attention, memory performance, physical performance, muscle recovery and intraocular pressure and a possible decrease in risk of heart disease.
Phytosterols: Phytosterols, a generic term that refers to both plant sterols and stanols, are a group of lipophilic steroid alcohols that naturally occur in plants. To date, more than 250 phytosterols have been identified and successfully isolated from various plants33. The best dietary sources of phytosterols are unrefined vegetable oils, seeds, cereals, nuts and legumes (sterols) and corn, wheat, rye and rice (stanols)34. Concerning the health benefits, phytosterols are reported to lower cholesterol33 and cancer protection35. It was proved that dietary supplementation with plant sterols, stanols and their esters reduces intestinal cholesterol absorption36. Meanwhile, studies have proved the evidence that phytosterols have a role in protecting against the development of various cancers (ovarian, breast, stomach, prostate and lung cancer)37. This has been attributed to the effect of phytosterols on the membrane structure and function of tumour and host tissue and stopping the growth and spread of cancer cells.
Thiosulfonates: Thiosulfonates are organic sulfur-containing compounds isolated from plants i.e., garlic, onion, cabbage and cauliflower. Propyl-propane thiosulfonate, isolated from garlic, affects enteropathogens, such as subspecies Salmonella, Campylobacter jejuni, Clostridium perfringens and Escherichia coli 38. Thiosulfonates reduce blood pressure and have anticancer and antimicrobial properties39. The antimicrobial activity of thiosulfate acids is closely related to their ability to block the normal metabolism of microorganisms through sulfenylation of thiol groups of their enzymes40. Moreover, organosulfur compounds found in garlic and onions are known, as anticarcinogenic agents. In this respect, Rao et al.41 mentioned that diallyl sulfide (DAS), a major component of garlic oil, is an inhibitor of tumorigenesis by various metabolically activated carcinogens.
In vitro production of plant nutraceuticals: The recent advances in biotechnology particularly methods of plant tissue cultures offer new means for the in vitro production of valuable plant phytochemicals. In this respect, different tissue culture techniques such as cell, callus and organ cultures have been developed as an alternative for the production of economically important valuable phytochemicals from field-grown plants. The greatest advantage of the plant tissue culture technique has been its ability to provide a continuous, sustainable, economical and viable production of natural compounds, regardless of the geo-climatic conditions and under a microenvironment regime highly controlled42. Moreover, cell suspension cultures and bioreactor techniques could be used to regulate metabolic processes to maximize yields43. Otherwise, hairy root cultures provide instrumental in enhancing the production of valuable plant metabolites44. For improving the production of phytochemicals of interest by plant cell cultures, different steps should be achieved: (1) Identification of plant starting materials that contain the largest amount of the desired compound, (2) Optimization of culture medium and culture conditions, (3) Addition precursors or elicitor to in vitro cultures that can drive and speed up the metabolic flow leading to the phytochemical of interest, (4) Selection of the most productive plant cell lines and (5) Scale-up through the use of appropriate bioreactors.
For improvement phytochemicals production, mutagenesis methods such as irradiation and chemical mutagens have been applied to induce mutations in plants. Mutagenesis of cultured explants, cells and tissue cultures represents a feasible method for induction of genetic variability. In vitro mutagenesis is a genotypic change in a culture and the derived population can be maintained via rigorous subculturing. This mutagenesis can be achieved by mutation-induced by radiations or chemicals. The advantages include low exposure levels and a wide variation of mutation. At the present, elicitation is the most effective biotechnological tool for enhanced biosynthesis as well as accumulation of nutraceuticals in vitro plant cultures. The application of elicitors reduced the period of cultivation and yielded a high level of product. In this respect, various types of elicitors have been used to modify plant cells’ metabolism to enhance the accumulation of desired compounds. However, various parameters, such as elicitor type, concentrations, duration of exposure, treatment schedule, culture type, cell line, medium composition, presence or absence of growth regulation and age or stage of the culture at the time of elicitor treatment are important factors that can determine the effectiveness of any elicitation strategy on plant nutraceuticals production45. Otherwise, genetic engineering can be used to manipulate the metabolic pathways of plant nutraceuticals. In this context, hairy root cultures have become an important method for producing secondary metabolites in plant tissues because of their stability and high productivity in hormone-free culture conditions46. Therefore, the productivities of hairy root cultures have been reported to be enhanced through adopting strategies in plant metabolite production enhancement47. Recently, nanoparticles have been extensively used in plant tissue culture to induce various growth parameters as well as bioactive metabolites improvement48. All of the studies mentioned above confirm the possibility of nanoparticles being employed as successful and promising bioactive compound elicitors in plant cell and organ cultures. Meanwhile, bioreactors are utilized in bioprocessing industries, wherein the optimum environmental conditions are maintained to achieve the required biological products on a large scale. The advantages of bioreactors include a better rate of product multiplication, lesser time for multiplication and a minimum cost49.
In vitro culture was found more efficient than the whole plant for the production of different bioactive secondary metabolites since the level of ajmalicine, ajmaline, anthraquinones, benzylisoquinoline alkaloids, berberine, bisoclaurine, coniferin, diosgenin, ginseng, ginsenoside, glutathione, nicotine, rosmarinic acid, raucaffricine, shikonin, taxol, turpentine, triptolide, ubiquinone-10, etc., were high in in vitro culture compared to whole plant production following agronomic method50. In this respect, callus induction of globe artichoke was achieved as a method of in vitro production of caffeoylquinic acids and phenolic compounds51. Likewise, the production of total phenolic and total flavonoids of chicory from adventitious root cultures was reported by Ibrahim et al.52. Callus cultures of Lepidium sativum L., were established for in vitro production of glucosinolate compounds53. Furthermore, different abiotic stresses were used for enhancement of in vitro production of phenolics54, anthraquinone55, flavonoids56 and anthocyanin57. On the other hand, fatty acids were enhanced in callus cultures of jojoba by gamma irradiation treatment58.
Enhancement of nutraceuticals production by mutation: The mutation is reported as an important factor in improving nutritional quality in plant species. Physical or chemical mutagenic agents are used to induce mutations in different planting materials. Regarding physical mutagens, ionizing radiations have been used widely for inducing hereditary aberrations and more than 70% of mutant varieties were developed using physical mutagenesis59. The major advantage of using physical mutagenesis compared to chemical mutagenesis is the degree of accuracy and sufficient reproducibility, particularly for gamma rays, which have a uniform penetrating power in the tissue60. In the mutation-breeding programme, in vitro cultured explants provide a wider choice of controlled selection following mutagenic treatment as compared to treatments of in vivo material. Moreover, in vitro culture techniques are particularly relevant for mutagenesis as totipotent plant cells are cultured proliferated in large volumes and can be induced into the regeneration of complete plants. Among the different in vitro methods, somatic embryogenesis is the most useful tool for mutagenesis as somatic embryos usually originate from single cells. Furthermore, haploid cell and protoplast cultures have advantages in studies on mutant selection in vitro, since mutations particularly recessive in nature can easily be detected in subsequent generations. In this respect, gamma rays are known to influence plant growth and development by inducing cytological, genetic, biochemical, physiological and morphogenetic changes in cells and tissue. The effects of gamma radiation on changes in plant photosynthetic pigments, the composition of chemical, cellular structure and crude oil yield were investigated61. To improve the oil content of jojoba, El-Shabrawi et al.58, exposed embryonic callus to different doses of gamma radiation. They found that oil content was increased by 1.41% by exposing it to 5 Kr gamma radiations. Increasing in ginsenoside production in calli cultures of Panax ginseng was also observed by exposure to gamma radiations (10-100 Gy)62,63. A dosage of 30 Gy was selected as the adequate dose and via HPLC and TLC analysis, it was confirmed that there was an increase in ginsenoside production in the mutant lines. To enhance phenolic compounds and flavonoids in Artemisia annua, callus cultures were exposed to different doses of gamma irradiation64. Irradiation dose of 15 Gy showed the highest amounts of phenols and flavonoid content as compared to the control. Likewise, the low doses (16 Gy) of gamma irradiation stimulated the production of shikonin derivatives in callus cultures of Lithospermum erythrorhizon but higher doses than 32 Gy did not enhance the shikonin derivatives65. Also, El-Beltagi et al.66 found that low doses of gamma irradiation (15 and 20 Gy) enhanced the total phenolic and flavonoid accumulation in rosemary (Rosmarinus officinalis L.) callus cultures. Similarly, a 15 Gy dose of gamma irradiation slightly enhanced stevioside content over the control of Stevia rebaudiana callus cultures67. Gamma irradiation was also used for promoting some phytochemicals such as ascorbate, photosynthetic pigments and some antioxidant enzymes activity in red pepper (Capsicum annuum) when treated with gamma rays ranging from 2-16 Gy68.
On the other hand, chemical mutagens are applied as an alternative to induce mutations where no physical is available. An advantage of chemical mutagenic agents is that they can be applied without complicated equipment or facilities. But, the chemical mutagens are extremely toxic compared with physical mutagens and require more care in their application. Chemical mutagens belonging to the class of alkylating agents are mostly used such as ethyl methanesulfonate (EMS), diethyl sulfate (dES), ethylamine (EI), ethyl nitroso urethane (ENU), ethyl nitrosourea (ENH) and methyl nitrosourea (MNH). Among these mutagens, EMS has been used in several crops such as urd bean69 and barley70. Mostafa71 stated that the efficiency of mutant induced by chemical mutagens is influenced by pH, soaking into water, temperature, concentration and treatment duration. Particularly, in vitro mutagenesis depends on the establishment of reproducible in vitro plant regeneration procedures, optimization of mutagenic treatments and screening of the mutagenized populations for desired variations. To develop reproducible protocol mutagenesis of Hyoscyamus niger targeting putrescine N-methyltransferase (PMT) and 6β-hydroxy hyoscyamine (H6H) genes of alkaloid biosynthetic pathway, callus was treated with different concentrations (0.01-0.1%) of ethyl methane sulfonate (EMS)72. The results depicted that EMS has an intense effect on PMT and H6H gene expression and metabolite accumulation. The mutagenic potential of EMS was also recorded on biochemical components, cytological features and morphology in Coriandrum sativum L.73. At this point, the induction of polyploidy or artificial chromosome doubling is implemented for enhancing the biomass of a plant and, thus consequently amplifying the metabolite profile as well. The superiority of secondary products of polyploidy compared to diploid might be the expression increasing of crucial biosynthesis genes in polyploidy. In this respect, colchicines have been widely used in increasing the level of ploidy. In this context, adventitious roots of Panax ginseng were treated with 100 mg L–1 colchicines over 60 hrs to obtain octoploid genotypes. HPLC analysis, of the resultant regenerated octoploid plantlets, demonstrated that the chromosome doubling can enhance biomass and ginsenoside accumulation, simultaneously74. Likewise, autotetraploid of Bacopa monnieri was obtained in vitro by exposure nodal segments to colchicine (0.5%) for 48 hrs75. The maximum total bacoside content was obtained from an autotetraploid plant, which was 2.3-fold higher than the level in diploid plants. Similarly, polyploidization enhances flavonoids in Fagopyrum tataricum as compared to diploid plants76.
Enhancement of nutraceuticals production by transformation: Transformation is another strategy that can be used to produce high-value-chemical products in plant cells or organ tissues. Manipulation of plant metabolic signalling pathways has been intensively used to enhance the production of valuable compounds in plants and in vitro systems77. The transformation was found to be useful in those cases where different methods commonly used to increase secondary metabolite production (cell selection, elicitor treatments and addition of a biosynthetic precursor) only slightly enhance cell productivity. Results of research prove that the transfer of some DNA (T-DNA) oncogenes, such as the rolB and rolC genes of Agrobacterium rhizogenes and the 6b gene of Agrobacterium tumefaciens, affect the biosynthesis of secondary metabolites in transformed plant cells78,79. Hairy root culture is the most promising transformation technique used for in vitro production of valuable plant compounds. Hairy roots are unique in their genetic and biosynthetic stability, faster in growth and more easily maintained. Furthermore, a hairy root can be transformed to produce more than one secondary metabolite and this unique ability makes them very economical for commercial exploitation80. These roots are induced on aseptic, wounded parts of plants by inoculating them with Agrobacterium rhizogenes. Sevón and Oksman-Caldentey81 mentioned that the secondary metabolites produced by hairy roots arising from the infection of plant material by Agrobacterium rhizogenes are the same as those usually synthesized in intact parent roots, with similar or higher yields. During the infection process, Agrobacterium rhizogenes transfers a part of the DNA (transferred DNA, T-DNA) located in the root-inducing plasmid Ri to plant cells and the genes contained in this segment are expressed in the same way as the endogenous genes of the plant cells. Moreover, the auxin content of the transformed root is comparatively higher than the non-transformed roots which play an important role in their growth behaviour82. In this context, several studies have demonstrated that Agrobacterium rhizogenes-mediated transformation with root locus (rol) genes enhances secondary metabolite biosynthesis in transgenic roots by activating biosynthetic genes79,83,84. Hairy root cultures of many plant species have been widely studied for the production of secondary metabolites useful as pharmaceuticals, cosmetics and food additives83,85,86. Furthermore, elicitation of hairy root promotes phytochemical production and also arrests feedback inhibition, preventing degradation of metabolites in the culture medium87. In this respect, Ismail et al.88, found that total flavonoid and phenolic contents were higher in hairy root cultures of Lactuca sativa as compared to the non-transformed ones. Hairy root cultures of Momordica charantia were elicited with jasmonic acid and salicylic acid to enhance biomass accumulation and phenolic compound production89. The rolB gene was introduced in Vitis amurensis cells and the rolB-transformed calli are able of producing up to 3.15% dry weight of resveratrol90. This ability is tightly correlated with the abundance of rolB mRNA transcripts. In their investigation of in vitro production of phenolic compounds from hairy root culture of Tartary buckwheat, Kim et al.91, found that the concentration of phenolic compounds in the hairy roots was several-fold higher compared with wild type roots of the same species.
Genetic engineering is used to improve minerals and vitamins such as carotenoids in food that can be useful in the treatment of some cancers, heart disease and blindness92. Also, genetic engineering can be used to increase the levels of unsaturated fatty acids in some commonly used oils such as canola, soybean, sunflower and peanuts93. Genetic modification leads to oilseed crops with unusual fatty acids such as short-, medium- and long-chain fatty acids and those with double bonds at unusual positions, or those that carry hydroxyl or epoxy groups. On this point, the strategies for enhancing the production of plant nutraceuticals in vitro are built on the manipulation of existing metabolic pathways by overexpressing or silencing selected elements involved in their biosynthesis. One of the successful examples of approaches to of enhancement plant nutraceuticals using genetic engineering is the development of “Golden Rice” involving the transfer of the genes necessary for the accumulation of carotenoids (vitamin A precursors) in the endosperm that are not available in the rice gene pool94-96. Potato tubers were biofortified by three bacterial genes encoding phytoene synthase, phytoene desaturase and lycopene β-cyclase to increase β-carotene97. The transformation of the PrLeg gene into the potato, which contains low amounts of sulfur-containing amino acids, was found to enhance Met content in the tubers98. In tomatoes, increased content of flavanols in fruits was achieved by engineering tomato plants with transcription factor (LC and C1) genes. The ectopic expression of these two genes allowed the upregulation of flavonoid pathway genes and the accumulation in berry flesh of these antioxidant compounds99. The increase of anthocyanins in “purple” tomato was achieved by expressing two genes of Antirrhinum majus en coding the transcription factors Delila and Rosea1100. Likewise, excessive expression of the Petunia CHI gene caused a 78-fold increase in flavonoids in tomato peel101. Shi et al.102 reported the development of canola transgenic with change in fatty acids compositions, using B. napus cultivar “CY2” as the transgenic recipient of BnFAE1, a fragment involved in the synthesis of very-long-chain fatty acids. Gold kiwifruit was genetically modified for a high level of ascorbic acid, carotenoids and lutein and zeaxanthin103. Vereshchagina et al.104 transformed artichoke with the rolC gene, which is a known inducer of secondary metabolism. Analysis revealed that the predominant metabolites synthesized in the transgenic calli were 1,5-dicaffeoylquinic acid, 3,4-dicaffeoylquinic acid and chlorogenic acid. The overall production of these metabolites was three times higher than that of the corresponding control calli. In another study, stilbene synthase genes isolated from Vitis vinifera were transferred to Lactuca sativa, resulting in transgenic red lettuce capable of producing a high amount of resveratrol105.
Enhancement of plant nutraceuticals production by elicitation: Elicitation is one of the most important strategies for enhancing the in vitro production of plant phytochemical compounds. Elicitors are stress factors that trigger the inducible defence changes in a plant system that results in induction or expansion of biosynthesis of fine chemicals106. Elicitors have been widely used to increase the production of plant metabolites in in vitro plant cell cultures. Hairy root cultures are preferred for the application of elicitation due to their genetic and biosynthetic stability and high growth rate in growth regulator-free media. Depending on their nature, elicitors are classified as biotic (biological origin) and abiotic (non-biological origin). Biotic elicitors include compounds released by microorganisms and other pathogens or formed by the action of plant enzymes on microbial cell walls, microbial enzymes, fungal and bacterial lysates, yeast extracts and polysaccharides from microorganism cell walls. On the other hand, chemical substances, e.g., mineral salts, heavy metals, or physical factors such as light (UV-B, UV-C radiation), temperature and osmotic stress represent abiotic elicitor’s factors. Several parameters such as elicitor concentration and selectivity and duration of elicitor exposure are influencing the successful elicitation. A high dosage of elicitor has been reported to induce hypersensitive response leading to cell death, whereas an optimum level was required for induction107. Otherwise, the different elicitors require a different duration of time to elicit the plant cell culture. Moreover, the composition of the medium or selection of medium also played a vital role in the elicitation process.
Enhancement by abiotic elicitors: The abiotic elicitors enhance the biosynthesis of plant secondary metabolites by triggering the defense- or stress-induced responses108. In this respect, Naik and Al-Khayri109 mentioned that abiotic elicitors have different effects on the plant cellular processes such as carbon partitioning, carbohydrate and lipid metabolism, osmotic homeostasis, protein synthesis and gene expression. Their effect depends on the concentration of the elicitor, the growth stage of the culture, the period of contact and the time course of the elicitation. The research results proved that salt stress increases terpenes, phenols and alkaloids compounds in plant cells110. Also, saltation of the culture medium with NaCl enhanced the total ginseng and saponin in hairy root cultures of Panax ginseng111. In response to salt stress, anthocyanins are reported to increase in the non-sensitive species112. In red peppers, total phenolic content increased with a moderately saline level in culture medium113. Hussein and Aqlan114 found that salt stress induced by the lower NaCl concentration increased total phenolics, total flavonoids and total tannins in callus cultures of Fenugreek (Trigonella foenum-graecum L.). Salinity also increased the diamine and polyamine content in Oryza sativa115. Likewise, light can enhance several metabolites in certain plants as a physical factor116-118. In this respect, anthocyanins and flavones increased in response to high visible light levels119. Similarly, UV–B irradiation stimulates flavonoid synthesis by enhancing the phenylpropanoid pathway in Kalanchoe pinnata120. Also, an increase in astragaloside biosynthesis was observed in hairy root cultures of Astragalus membranaceus when exposed to UV-B121. Yu et al.122 studied the effect of different light sources on both biomass and ginsenosides biosynthesis in ginseng hairy root cultures and they found root growth was stimulated by red light in comparison to dark treatment. These results suggest that it is possible to manipulate secondary metabolite accumulation by varying the light and dark regimes.
In terms of chemicals elicitation, salicylic acid and jasmonic acid have been used to induce the production of phenolic compounds by in vitro cultures, being signalling molecules of different pathways123. When exogenously applied to plant cell cultures of a variety of species, methyl jasmonate positively stimulates the workflow of secondary biosynthetic pathways, leading to increased production of terpenoids, flavonoids and alkaloids and phenylpropanoids124,125. Jasmonic acid was used for enhancing the phenolic compound production and flavonoid contents in hairy root cultures of Momordica charantia (Cucurbitaceae)92. Salicylic acid (SA) also affects nutraceuticals metabolism in plants. It induced accumulation of the triterpenoids ginsenosides in ginseng and glycyrrhizin in liquorice126,127. Evidence demonstrated that suitable concentrations of SA can also promote monoterpene production128. Likewise, salicylic acid exhibits improving anthocyanin content in calli and cell cultures of Vitis vinifera L. cv. Gamay Fréaux123,129. Capsaicin was improved in suspension cultures of Capsicum frutescens by supplying isocaproic acid as a precursor130. Regarding the application of hormonal compounds, the effect of different concentrations of ethephon on cynarin accumulation in callus cultures of globe artichoke was evaluated131. High cynarin content of the callus cultures was observed by the addition of 90 μL ethephon into the culture medium for 9 and 12 days. On the other hand, metals like Ni, Ag, Fe and Co have been shown to elicit the production of secondary metabolites in several plants. For instance, copper was used to enhancing the production of phenolic compounds in the root culture of Panax ginseng, increasing up to 76% of the production of phenolics and flavonoids126. Increasing four times more anthocyanin accumulation in the cell culture of Vitis vinifera cv., Gamay Red was obtained by the application of magnesium to the culture medium132. The combination of CuSO4 and Dimethyl Sulfoxide (DMSO) increase the grindelic acid production in callus and cell suspension cultures of Grindelia pulchella133.
Enhancement by biotic elicitors: Biotic elicitors are extracts or products derived from biological origins such as fungal, bacterial, yeast or plant. The most commonly biotic elicitors are polysaccharides, glycoproteins, inactivated enzymes, chitosan, pectin, chitin, alginate, curdlan, xanthan, elicitin134. In this respect, chitosan the biotic elicitor polysaccharide was used to improve the yield of many useful compounds in tissues and whole plants. The effectiveness of polysaccharides, among which chitosan, as active elicitors, is dependent on their molecular structures. In general, polysaccharides with a high number of side chains are more active than those with a lower number. Chitosan treatments have been shown to act as an elicitor in plants, to enhance phytochemical contents of cells and tissues of different varieties135,136. In the basil plant, chitosan increased the total amount of phenolic and terpenic compounds, especially rosmarinic acid and eugenol which have strong antioxidant, antiviral, antibacterial, anti-inflammatory, antiseptic and anaesthetic properties and show application in perfumery and flavouring137. High levels of alkaloids coumarins and fluoroquinolone were found in shoot cultures of Ruta graveolens by supplementation of culture medium with chitin or chitosan138. In a study on elicitation of Morinda citrifolia plant, Purwianingsih et al.139, found that the addition of chitosan into medium amended with 2,4-D and kinetin increases anthraquinone in callus cultures. Likewise, chitosan enhanced the production of trans-resveratrol and vinifera in the cell system of V. vinifera140. On the other hand, Aspergillus niger is used as a fungal elicitor to enhance the production of valuable compounds in plants. The positive influence of Aspergillus niger in combination with methyl-jasmonate on in vitro aggregation of total phenolic and peroxides from date palm was reported by Taha et al.43. Also, augmentation of culture medium with 1.5% of Aspergillus niger in combination with 100 μM of methyl jasmonate increased capsaicin accumulation in Capsicum annum cell culture141. Cai et al.142 reported that the number of flavonoids in the Avena sativa L. was markedly increased by the fermentation by the Aspergillus niger. In this context, different concentrations of a root endophytic fungus Piriformospora indica cell homogenates were utilized as an abiotic elicitor in Withania somnifera hairy root for different periods143. When the hairy root cultures of Withania somnifera were treated with 3% cell homogenates of Piriformospora indica for 48 hrs, it enhanced the biomass and the production of withanolides, viz., withaferin A, withanolide A, withanoside V and withanoside IV as compared to control (untreated hairy root).
Elicitation by nanoparticles: The application of nanotechnology is a new sustainable generation of in vitro cultures for the improvement of the plant’s active constituent production. Nanoparticles have the promise to be used as novel effective elicitors in plant biotechnology. In this context, the elicitation of nanoparticles was found to increase the amounts of bioactive compounds accumulated in various plants144-150. The effects of metal oxide nanoparticles such as: titanium oxide, zinc oxide, iron oxide and copper oxide have been indicated for facilitating plant growth and production of secondary metabolites151,152. The total phenols and flavonoid contents of Cucumis anguria hairy root cultures were significantly increased by silver nanoparticles elicitation153. Likewise, zinc oxide nanoparticles have an obvious effect on the accumulation of plant secondary metabolites such as flavonoids and phenols. The influence of copper oxide nanoparticles (CuO NPs) on the aggregation of glucosinolates and phenolic compounds in hairy root cultures of Chinese cabbage was investigated154. Glucosinolates (gluconasturtiin, glucobrassicin, 4-methoxyglucobrassicin, neoglucobrassicin, 4-hydroxyglucobrassicin, glucoallysin, glucobrassicanapin, sinigrin, progoitrin and gluconapin) and transcript (MYB34, MYB122, MYB28 and MYB29 ) levels were considerably escalated in CuO NPs-elicited hairy root cultures compared to non-elicited cultures. Moreover, phenolic compounds (flavonols, hydroxybenzoic and hydroxycinnamic acids) were significantly enriched in CuO NPs-elicited hairy root cultures. The presence of TiO2 nanoparticles (4.5 or 6.0 mg L–1) significantly increased the content of gallic acid, chlorogenic acid, o-coumaric acid, tannic acid and cinnamic acid in embryonic calli of Cicer arietinum151. The accumulation of steviol glycosides in shoot cultures of Stevia rebaudiana was significantly enhanced on MS medium fortified with 1 mg L–1 CuO nanoparticles149. In addition, the total flavonoid and phenolic content also increased with ZnO nanoparticles treatment. However, higher concentrations of ZnO nanoparticles led to decreased secondary metabolite production due to the phytotoxic effects of ZnO nanoparticles. Concerning Ag NPs, Bhat and Bhat155 found that 3 mg L–1 to Capsicum frutescens cell cultures, increased the content of capsaicin about 2-fold.
Scaling up and using of bioreactor for production of plant nutraceuticals: With the increasing demand for nutraceuticals derived from plants, in vitro culture became a reliable technique for the mass production of such compounds. In practice, in vitro cultures provide a source of homogeneous highly active cells that allow overcoming some plant limits such as the slow growth, seasonal and environmental variations and disease susceptibility. Adventitious roots or non-transgenic roots can be utilized for producing plant natural products, especially those that are linked to root differentiation. Moreover, the cell culture technique could be used for the large-scale production of desired compounds from different plant species. In this respect, a bioreactor system is employed for the large-scale production of valuable bioactive compounds from plant cells. Many physical factors, like the intensity, stress and operation conditions should be
optimized as a first step in scaling up the process through bioreactors. The scaling-up from shake flasks to bioreactors is typically not straightforward due to changes in the cell growth environment. By improving bioreactor design and optimizing key parameters of the culture, some of these limitations can be overcome. In this context, different varieties either old or new designs of bioreactors are used for large-scale culturing of plant cells or other microorganisms156-158. Due to the easy scaling-up, good fluid mixing and oxygen transferability, the conventional stirred tank reactor (STR) bioreactor is most widely exploited. Moreover, this type of bioreactor is recommended for optimal production of the target Phyto molecule by elicitation159. For the large-scale harvesting of phytochemicals, it is possible to use two kinds of procedures. When the target compound accumulates inside the cells, the final biomass is harvested from the tank of the bioreactor and the target compound is extracted from the cells. On the contrary, when the product is released from the plant cells, part of the culture medium is removed from the tank and replaced by the fresh medium to rejuvenate the cell biomass.
Scaling-up of hairy roots and their phytochemical compounds to an industrial scale using bioreactors has developed160,161. In this respect, a bioreactor system has been utilized for the production of different compounds from cell and hairy root cultures of many species. Enhancing ginsenoside production in adventitious/hairy roots of ginseng in various levels of liquid-phase airlift bioreactors was achieved by optimizing nutrient growth and elicitors162. The accumulation of phenolic compounds: Phenolic acids and flavonoids in the micro shoots of Schisandra chinensis grown in different types of the bioreactor was evaluated163. The maximum phenolic acids content was recorded in the biomass maintained in the cone-type bioreactor for 30 days. While the highest total content of flavonoids was found in the micro shoots maintained in the nutrient sprinkle bioreactor for 30 days. Large-scale production of steviol glycosides (low-calorie glucoside sweeteners) in Stevia rebaudiana cultures was achieved in a temporary immersion bioreactor164. In vitro aggregation of phenolic and peroxides compounds from cells of date, palm cv. Zaghlool using stirred tank reactor was reported43. The maximum content of these compounds was obtained by elicitation of modified MS-medium with Aspergillus niger extract at 0.1% combined with methyl-jasmonate (100 μM), after 10 days of cultivation.
Plants are considered biochemical factories to produce nutraceuticals such as alkaloids, flavonoids and polyphenols. In vitro culture technique, in which plant cells, tissues and organs offer alternatives for producing the important plant nutraceuticals. Moreover, biotechnological approaches such as mutagenesis, transformation and elicitation can be employed to enhance the nutraceuticals production utilizing in vitro techniques. Although it needs complicated facilities, physical mutagenesis produces sufficient reproducibility compared to chemical mutagenic agents. On the other hand, transformation using Agrobacterium rhizogenes is used to enhance nutraceuticals in plant cells or organ tissues.
Meanwhile, elicitation is widely used to increase the biothensis of plant metabolites in plant cell cultures. The elicitor’s effects depend on the concentration of the elicitor, the growth stage of the culture and the exposure period. On the other hand, a bioreactor system has been developed for the large-scale production of desired compounds from plant cell suspension and organ cultures.
CONCLUSION
Plant tissue culture techniques offer a perpetual source for the continuous production of plant nutraceuticals under controlled conditions. Moreover, mutagenesis, transformation and elicitation can be used to enhance the plant nutraceuticals production. To induce mutations, planting materials are exposed to physical i.e., irradiation or chemical mutagenic agents such as methanesulfonate, diethyl sulfate, ethylamine, ethyl nitroso urethane ethyl nitrosourea and methyl nitrosourea. Transformation can be used for the manipulation of plant metabolic pathways leading to enhancing the production of valuable compounds in plants. In this respect, hairy root culture induced by Agrobacterium rhizogenes was found the most promising transformation technique used for in vitro enhanced production of valuable plant compounds since the rolB and rolC genes of Agrobacterium rhizogenes positively affect the biosynthesis of secondary metabolites in transformed plant cells. Otherwise, biotic and abiotic elicitors enhance the plant nutraceuticals by acting on their biosynthesis enzymes. On the other hand, different designs of bioreactors can be used for the large-scale production of nutraceuticals from plant cells. The conventional stirred tank reactor (STR) is the most common bioreactor used for plant cells.
SIGNIFICANCE STATEMENT
Although the healthcare has advanced techniques to treat different diseases, still nutraceuticals represent great importance because of the power it holds to treat diseases through diet. This review is focused on the various biotechnological approaches used for the production of plant nutraceuticals as well as their contribution to public health. In this respect, various techniques of in vitro culture have been used for the production of plant nutraceuticals under aspects conditions. The main advantage of in vitro culture over the conventional cultivation of whole plants is that nutraceuticals can be produced under controlled conditions independent of climatic changes. In this regard, the cell culture technique in combination with elicitation now represents an efficient way to produce the plant nutraceuticals in suspension cultures. Otherwise, hairy roots cultures, induced by infection of Agrobacterium rhizogenes proved to be an important technique not only for in vitro production but also for biosynthesis enhancement of such plant compounds. Meanwhile, such compounds can be commercially manufactured using a bioreactor which offers the opportunity for large amounts of production with the maintenance of genetic integrity of plant cell lines. Generally, this article will help to recognize an applicable method of in vitro production of plant nutraceuticals using different types of techniques and enhancement factors.