In vitro strategies for the enhancement of secondary metabolite production in plants: a review
Bulletin of the National Research Centre volume 46, Article number: 35 (2022)
Plants are the prime source of vital secondary metabolites (SMs) which are medicinally important for drug development, and these secondary metabolites are often used by plants in the various important tasks like defense against herbivory, interspecies defenses and against different types of stresses. For humans, these secondary metabolites are important as medicines, pigments, flavorings and drugs. Because most of the pharmaceutical industries are highly dependent on medicinal plants and their extraction, these medicinal plants are getting endangered.
Plant cell culture technologies are introduced as a viable mechanism for producing and studying SMs of plants. Various types of in vitro strategies (elicitation, hairy root culture system, suspension culture system, etc.) have been considerably used for the improvement of the production of SMs of plants. For the enhancement of SM production, suspension culture and elicitation are mainly used, but hairy root culture and other organ cultures are proved to satisfy the demand of secondary metabolites. Now, it is easy to control and manipulate the pathways that produce the plant secondary metabolites.
Techniques like plant cell, tissue and organ cultures provide a valuable method for the production of medicinally significant SMs. In recent years, most of the in vitro strategies are used due to knowledge and regulation of SM pathway in commercially valuable plants. In future, these things will provide a valuable method to sustain the feasibility of medicinal plants as the renewable sources of medicinally important compounds, and these methods will provide successful production of desired, important, valuable and also unknown compounds.
Plants are the valuable source of medicines that play a vital role in the mitigation of overall global health issues (Constabel 1990). Since from ancient times, humans’ race have relied on the traditional medicinal plants. Most of the plants synthesize large number of organic compounds which do not directly participate in the growth and development of the plant but play a significant role in plant–plant, plant–environment interaction or defensive role. These substances are called ‘secondary metabolites’ (Hussain et al. 2012). These metabolites do not participate in plant growth and development so they are produced in low quantity (Kim et al. 2002b). Secondary metabolites are used as agrochemicals, pharmaceuticals, flavor, fragrance, food additives and pesticides (Balandrin and Klocke 1988). Besides, they are performing a potent role in fighting COVID-19 (Khan et al. 2021; Hema et al. 2020). There are various roles of secondary metabolites such as protection of plants against herbivores and microbes, attracting chemicals for allelopathic agents (chemicals that influence competition among plant species), pollinators and seed-dispersing animals (Rodney et al. 2000). These are different from primary metabolites. Primary metabolites are metabolic intermediates, essential for growth and development, participated in important metabolic processes such as respiration and photosynthesis while SMs are derivatives of primary metabolites. Synthesis of primary metabolites takes place from three metabolic pathways: the Embden–Meyerhof–Parnas (EMP) pathway, hexose monophosphate (HMP) pathway and the Entner–Doudoroff pathway. SMs are synthesized either by the malonate/acetate pathway or the shikimic acid pathway (Hussain et al. 2012). The medicinal plants are getting endangered because most of the pharmaceutical industries are highly dependent on medicinal plants and their extraction. Due to their complex structure, it is difficult to synthesize these organic compounds chemically and through conventional methods. Therefore, in vitro method for production of plants SMs is the sustainable way to achieve the market demand. Besides this, plant tissue culture technique is an efficient and best alternative solution to the difficulties faced by the phytopharmaceutical industries in particular for mass propagation, germplasm conservation, study and production of biologically active compounds and for improvement of genetics. In vitro plant materials are valuable sources for the secondary metabolites production and also offer an exceptional environment for comprehensive investigation of biochemical and metabolic pathways (Mulabagal and Tsay 2004). In vitro regenerated plants provide uniform, sterile and compatible plant material. The plant materials are used in biochemical characterization and to distinguish the bioactive compounds. These compounds extracted from tissue cultures are easily purified due to uncomplicated extraction processes and absence of remarkable quantities of pigments, which probably reduce the production and processing costs. Because of these significant advances, research in this area has bloomed beyond expectations.
As plant cell, tissue and organ cultures are the sustainable ways to produce the useful secondary metabolites, but most of the experiments fail to produce the desired products. However, in many plant species, secondary metabolites production is enhanced by the callus cultures treated with elicitors, viz. salicylic acid, methyl jasmonate, chitosan and heavy metals (DiCosmo and Misawa 1985). In some plant species, secondary metabolites are also produced by hairy root culture and shooty teratoma (tumor-like) cultures. Generally, it is said that high levels of alkaloids are produced by hairy root culture (Sevon and Oksman-Caldentey 2002), whereas monoterpenes are produced by shooty teratomas (Spencer et al. 1993). However, there are a few examples of successful production of highly valuable SMs, using plant cells as factories. These include production of berberine by plant cell cultures of Coptis japonica and production of shikonin by cell suspension cultures of Lithospermum erythrorhizon (Fujita and Tabata 1986).
Field cultivation of plants to produce secondary metabolites has various difficulties (for example, low yields, instabilities in concentrations due to geographical, seasonal and environmental variations). This is why plant cell, tissue and organ cultures have been proved an appropriate alternative for the SMs production (Rao and Ravishankar 2002). Currently, different strategies have been established for synthesis of secondary compounds and the biomass accumulation such as improvement of strains, elicitation, permeabilization, optimization of media and culture environments, feeding of nutrients and precursors, biotransformation and immobilization methods. Also, in vitro studies including plant tissue culture and suspension cultures are used in different areas for the commercial production of SM (Ghorpade et al. 2011). SM production in large number has already been described in many plant cell suspension cultures (e.g., Berberis willsoniae, Coleus blumei, Coptis japonica and Lithospermum erythrorhizon), but some plant species fail to produce significant amounts of secondary metabolites in suspension culture (e.g., Atropa belladonna, Duboisia leichhardtii, Cinchona ledgeriana, Digitalis lanata) (Ellis 1988).
Enhancement of the secondary metabolites through plant cell culture
Plant cell and tissue cultures are another possible industrial method for the secondary metabolites production (Dicosmo and Misawa 1995). This method is self-sufficient and does not dependent on geographical or seasonal variation and accomplished by modification of different growth parameters. The idea that plant cell, tissue and organ cultures are an efficient method for the production of SMs, was introduced in late 1960. Different approaches were performed using cell culture system for the extensive production of SMs. Plant cell cultures produce SMs in different amounts and different qualities with respect to mother plants and these qualities may change with time (Tepe and Sokmen 2007). Cells are isolated from whole plant in plant cell culture, are cultured in suitable conditions and desirable product is drawn out from the cells which is cultivated. The advancements in plant tissue culture techniques provide excellent way to improve the secondary metabolites production (Chattopadhyay et al. 2002). Now, plant cell cultures play significant role in commercial production of SMs and also have advantages in cell biology, genetics and biochemistry research.
Cell suspension culture systems are instant method for industrial application and extensive production of SMs than tissue and organ culture. This method is an ultimate and reliable source for the production of natural products (Chattopadhyay et al. 2002; Vanisree et al. 2004). It is well known that many efforts have been made for commercial production of suspension culture. In suspension cultures, required metabolites are increased, but after some time, the synthesizing capacity decreases due to insufficient nutrition or genetic dissimilarities. So, selection and preservation of high yielding cell line is very important for suspension culture (Chattopadhyay et al. 2002).
Initially, calli are generated from selected mother plants in appropriate medium which is best suited for cultivation. This appropriate medium is helpful for dedifferentiation and differentiation mechanisms. However, this task is very experiential and critical to perform, but it could be done by other way like incomplete factorial experiments or surface response methods (Hamburger and Hostettmann 1991). These generated calli are subcultured either for propagation or to induced organogenesis, embryogenesis and suspension culture (Barrales et al. 2019). For the development of suspension culture, friable part of callus transferred into liquid medium which is maintained under appropriate environments of light, temperature, agitation, aeration and other physiological parameters. Different strategies are used to develop fairly homogeneous suspension culture. Various observations show that cells in suspension cultures are highly dependent on medium combinations, callus quality, genetic variation, etc. (Chattopadhyay et al. 2002). This technique has been successfully employed in several plant species (Table 1).
An elicitor is a substance which initiates or improves the biosynthesis of specific compound. Many traditional approaches are proved satisfactory to increase the SMs production, but elicitation is generally one of the utmost efficacious methods. Elicitors are used in very small concentrations to a living cell system which either induces or enhances the biosynthesis of SMs (Radman et al. 2003). Elicitors can be classified according to their ‘nature’ and the ‘origin’. According to their nature, elicitors are abiotic elicitors as well as biotic elicitors, while according to their origin, elicitors are exogenous elicitors as well as endogenous elicitors. Abiotic elicitors are the non-biological substances, mostly salts of inorganic compounds and physical factors such as Cu and Cd ions, Ca2+ and high pH conditions, whereas biotic elicitors are biological materials. Biotic elicitors include polysaccharides (pectin, cellulose, chitin, glucans), glycoproteins, G-protein and intracellular proteins. Exogenous elicitors are those compounds which are formed exterior to the cell, e.g., polysaccharides, polyamines and fatty acids, whereas ‘endogenous elicitors’ are those types of compounds which are formed inside the cell, e.g., galacturonide or hepta-β-glucosides, etc. (Veersham 2004).
In plant tissue culture, elicitation is also done in cell suspension cultures by applying chemical or physical stresses. Stresses trigger those secondary metabolites production that are normally not formed in that plants. Now, elicitation is done with biotic elicitors (chitosan, various protein extracts, sterilized mycelium of pathogenic fungi) and abiotic factors (heavy metal salts, high and low temperature, pH, UV light, etc. Many reports are available which show that elicitors increase the quantity of SMs in plant tissue culture (Tables 2, 3). Researchers, from all over the world, have applied various types of elicitors for the improvement of SMs production in in vitro system (Sudha and Ravishankar 2003; Karuppusamy 2009). Ahmad et al. 2019 used two elicitors, chitosan and yeast extract to examine the effects on 2-hydroxy-4-methoxybenzaldehyde (2H4MB), total phenolic content (TPC), total flavonoid content (TFC) and antioxidant activity in cell suspension culture of Decalepis salicifolia. Chitosan was found most effective to the yeast extract at 200 µM CH and 72 h of the incubation period. It increases the1.4-fold 2H4MB in relative to control, i.e., 10.8 µg/g. Maximum content of TPC and TFC was also found, i.e., 4.8 mg/g and 4.0 mg/g, respectively. Chodisetti et al. (2013) reported positive response of Aspergillus niger, Saccharomyces cerevisiae, Agrobacterium rhizogenes, Bacillus subtilis and Escherichia coli extracts in the gymnemic acid production, a SM obtained from Gymnema sylvestre. In the cultures of plant, gymnemic acid accumulation was in the order of A. niger, S. cerevisiae, A. rhizogenes, B. subtilis and E. coli. Putalun et al. (2010) used other elicitors in various concentrations like methyl jasmonate 50 μM, yeast extract 0.5 mg/l and chitosan 100 mg/l for stimulation of plumbagin production in Drosera burmanii. The result showed that yeast extract was the most effective to enhance the plumbagin production in roots that was 3.5-fold higher than control plants. At the same way, methyl jasmonate and chitosan also gave the highest concentration in shoot and root.
However, elicitation is very efficient method for enhancement of secondary metabolites but it cannot be sightlessly applied at any plant to induce metabolite production. Generally, metabolites induced or triggered by elicitation entail in plant defense system (Singh 1990). However, failure in triggering the production of SMs does not mean that the culture lacks metabolites rather continuous effects and screening are to be made. Those elicitors which are not specific to a species are not used for production of secondary metabolites because they induce ineffective elicitation. It was reported that an elicitor obtained from yeast extract could not trigger the phenylpropanoid pathway in cell suspensions of Vanilla planifolia (Funk and Brodelius 1990). But the same elicitor triggered the production of phytoalexin in Glycine max culture and alkaloid in Thalictrum rugosum and Eschschottzia californica. The pathway of phenylpropanoid could also be induced by using chitosan as an elicitor. Thus, the elicitation is an effective but challenging method and needs concentrated trial and error procedures (Singh 1990).
Enhancement of secondary metabolites through organ culture
It is clearly known that the secondary metabolites production through cell suspension culture is not always an adequate method; so, another method which is organ culture method uses as a supernumerary method for the SMs production (Giri and Narasu 2000; Verpoorte et al. 2002; Murthy et al. 2008; Baque et al. 2012).
Hairy root culture
Stewart et al. (1900) firstly introduced the term ‘hairy root’ in their literature. Hairy root cultures become an advance way in the field of plant tissue and organ culture for the enhancement of SMs production. This is done by transformation of required plant species by means of Agrobacterium rhizogenes, natural vector system. Agrobacterium rhizogenes causes hairy root disease in dicotyledonous plant (Giri and Narasu 2000; Bourgaud et al. 2001). A. rhizogenes, the gram-negative soil bacterium, uses dicotyledonous plant machinery for production of its own food source (opines) for which it transformed its genes in plant’s genome. This transformation causes the development of hairy root at the infection site of host plant (Shanks and Morgan 1999). Ackermann 1977 first time used A. rhizogenes mediated direct transformation in higher plants. Agrobacterium rhizogenes and plants comprise a complex sequence for interaction which are involved in A. tumefaciens.
Wounded plant cells released some phenolic compounds, viz. acetosyringone and α-hydroxy acetosyringone which recognizes by Agrobacterium as signal molecules and attached to plants’ cell (chemotactic response). Once bacterium attached or colonized to the wounded plant cells, it leads to the insertion of T-DNA fragment of Ti-plasmid (in case of A. tumefaciens) or Ri-plasmid (in case of A. rhizogenes) to the host plant cells and integrated into the plant genome. Many vir genes located in 40-kb region of Ti-plasmid or Ri-plasmid called the virulence (vir) region play significant characters in the Agrobacterium-mediated transformation process. The roles of Vir have been considered in a number of outstanding review articles (Christie 1997; Gelvin 2000; Tzfira et al. 2000; Zupan et al. 2000; Tzfira and Citovsky 2000, 2002). At the site of infection, hairy root tissues or neoplastic crown gall tumor are formed by genes of T-DNA fragment. Opines synthesis facilitate the hairy root formation. Bacteria used these opines as carbon and nitrogen source to invade in the plants (Binns and Thomashow 1988). The T-DNA encoded genes are enabled to express in infected plant cells because of having eukaryotic regulatory sequences. These events of transformation are activated by vir genes, which expressed only in the presence of acetosyringone. High level of vir gene expression is induced by various sugars which act as collegial with acetosyringone. Root formation takes place at the site of infection. At the infection site, the roots development takes place as the result of T-DNA genes which coded for synthesis of auxin and other rhizogenic functions. Mostly Agrobacterium strains hold single type of T-DNA, but some Ri-plasmids (carrier of agropine) contain two autonomous T-DNA represented as TL-DNA (left-handed T-DNA) and TR (right-handed T-DNA). Both the TR-DNA and TL-DNA have high resemblance to the T-DNA of the Ti-plasmid of A. tumefaciens and Ri-plasmid of A. rhizogenes strains, respectively (Nilsson and Olsson 1997). The transformation and integration of TL-DNA and TR-DNA takes place separately into the genome of host plant. It was previously known that auxin synthesis encoded by the TR-DNA and TL-DNA is responsible for synthesis of a compound that persuades the infected cells to discriminate into roots under the control of endogenous auxin synthesis (Ooms et al. 1986; Shen et al. 1988). Now, it is understood that the transference of TL-DNA is important for initiation of hairy root disease while TR- DNA transfer does not incite the roots development from the transformed cultures (Nilsson and Olsson 1997; Sevon and Oksman-Caldentey 2002). The transformation ability varies related to different strains of A. rhizogenes (Giri et al. 1997; Kumar et al.1991). It is well known that hairy roots formed by various bacterial strains show various morphologies and virulence. These variations could be described by the various plasmid concealed by the strain (Nguyen et al. 1992).
A. rhizogenes strains were classified into two groups, according to synthesis of opine by hairy roots (Petit et al. 1983):
Agropine-type strains (e.g., A4, 15,834, HR1, LBA 9402) induce roots to produce agropine, mannopine and corresponding acids.
Mannopine-type strains (e.g., 8196, TR7, TR101) elicit roots containing only mannopine, mannopinic acid and agropinic acid.
However, Zhou et al. (1997) classified the strains of A. rhizogenes into five classes: octopine, agropine, nopaline, mannopine and cucumopine.
In plant tissue culture, hairy root culture exhibits the greatest advantage for biosynthetic capacity of SMs production related to their mother plants (Kim et al. 2002a, b; Hao et al. 2020). Hairy root cultures accomplished unlimited growth in growth hormones free culture media. It is also acknowledged that hairy root culture facilitates the production of that SM which are not present in mother plant (Veersham 2004). There are many reports which show that hairy root cultures have been established in a number of dicotyledonous and monocotyledonous for production of SMs (Mukundan et al. 1997; Doran 2002; Rudrappa et al. 2005) (Table 4). If a specific SM accumulates only in the exposed part of the plant, in that case hairy root cultures accumulate the same SM (Wallaart et al. 1999).
Furthermore, transformed roots have the ability to form whole viable plants and sustain their genomic stability throughout continual subculturing and plant regeneration. Additionally, a transgenic root system has incredible possibility for integrating supplementary genes along with the Ri plasmid into the host plant cells. Hairy root cultures become an appreciated means for studying the biological qualities, properties and gene expression profile of metabolic pathways. Likewise, hairy root cultures are also applied for elucidation of the intermediates and crucial enzymes participated in the biosynthesis of SM (Hu and Du 2006).
Shoot culture is also an important method for the SMs production as hairy root cultures. It is done either to infect the aerial parts of the plants with Agrobacterium tumefaciens which lead to the formation of transgenic shoot (shooty teratomas) (Massot et al. 2000) or non-transgenic by the use of sufficient hormonal concentration (Saito et al. 1985). Some researchers proved that shoot cultures show nearly similar properties to hairy root cultures in production of SMs, genetic stability and relationship between growth and SMs production (Bhadra et al. 1998; Massot et al. 2000). But some differences are also existing in metabolites synthesis due to enzymes which are specially located either into roots or shoots (Subroto et al. 1996). The shooty teratomas are formed by transformation of the plant’s genetic materials with Agrobacterium tumefaciens nopaline or integration of A. tumefaciens Ti plasmid into the plant genome. Although the mechanism responsible for formation of shooty teratomas is not known, large number of plant species show the shooty teratomas formation (Hamill and Rhodes 1993). Limited number of reports are available related to growth, regeneration and application of shooty teratomas. Mainly shooty teratomas are applied in biotransformation. Saito et al. (1985) used shooty teratom as for nicotine biotransformation in Nicotiana tabacum. Shooty teratomas were produced in Mentha citrate to prove the existence of substantial amount of terpenes of mint oil (Spencer et al. 1990). In other work, Saito et al. (1991) observed that Atropa belladonna, N. tabacum and Solarium tuberosum synthesized tropane, nicotine and steroidal alkaloids, respectively, in shooty teratomas of. A poisonous alkaloid chemical compound, Solasodine in shooty teratomas of Solanum eleagnifolium, was reported by Alvarez et al. (1994). In some other plants, shooty teratomas are produced for secondary metabolites production, namely vincristine in Catharanthus roseus G. (Begum et al. 2009; Begum 2011), naphthoquinone in Drosera capensis var. alba (Krolicka et al. 2010).
Enhancement of secondary metabolites through biotransformation
As it is already known that plant cell suspension cultures are valuable method for the production of important SMs (DiCosmo and Misawa 1995; Kreis and Reinhard 1989; Longo and Sanroman 2006; Smetanska 2008). But, many of the trial of plant cell suspension cultures are unable to produce the desired SMs (Kreis and Reinhard 1989; DiCosmo and Misawa 1995; Vasil et al. 1984). However, these types of cell cultures are capable to convert the exogenous compounds in a different compound with new qualities through the processes of biotransformation. Biotransformation is a process in which the main substrates are transformed in a different substrate with new characteristics through the action of suitable enzymes or microorganisms (Ye et al. 2002, 2004, 2005; Ye and Guo 2005; Zhang et al. 2007; Zhao et al. 2007). Enzymatic potential of plant cells is helpful in the process of biotransformation. These enzymes have the capacity to catalyze the various reactions like regio- and stereoselective, hydroxylation, oxido-reduction, hydrogenation, glycosylation and hydrolysis of different organic compounds as well as microorganisms (Giri et al. 2001). This is because of plant enzymes are considered as important substance for the production of specific metabolites or different compound with new qualities (Ishihara et al. 2003). Biotransformation is differing from chemical methods because there is no need for the protection of labile functional groups (Simeo and Sinisterra 2009). Biotransformation process is done in many plant species like Eucalyptus perriniana in which thymol, carvacrol and eugenol converted into glycosides (Shimoda et al. 2006), in tobacco hyoscyamine converted into scopolamine by the process of biotransformation (Moyano et al. 2007) and in the Catharanthus roseus, glycosylation biotransformation of capsaicin and 8-nordihydrocapsaicin takes place in cell cultures (Shimoda et al. 2007). Therefore, biotransformation is a technique to synthesize new active components with different qualities. Biotransformation in the cell suspension cultures of Catharanthus roseus and Platycodon grandiflorum leads to the formation of a new compound as 1b-hydroxyl desacetylcinobufagin and other unknown compound which showed cytotoxic activities against HL-60 cell lines (Ye et al. 2003).The immobilised cell techonolgy has been used in the production of secondary metabolites (Yeoman 1987). The immobilised cell techonolgy has been used in the production of secondary metabolites (Yeoman 1987).
Role of genetic engineering in secondary metabolite production
Genetic engineering has a prominent effect on the accumulation of secondary metabolites. Plants are capable of producing a number of chemical compounds. However, these compounds serve specific functions in the plant, and has also effects on the human body, often with positive action against diseases. Over the years, natural products from plants and their non-natural derivatives have shown to be active against different types of chronic diseases. However, isolation of such natural products can be limited due to their low bioavailability and environmental restrictions. In vitro reconstruction of plant metabolic pathways and the genetic engineering of microbes and plants have been used to generate number of secondary metabolites (Vagner and Luzia 2014). Significant advances have been made through genetic engineering of microbes and plant cells to generate a variety of compounds (e.g., isoprenoids, flavonoids or stilbenes) using a diverse array of methods to optimize these processes. These approaches have been used also to generate non-natural analogues with different bioactivities. In vitro biosynthesis allows the synthesis of intermediates as well as final products (Siebert et al. 1996, Benedito et al. 2014).
Micropropagation in secondary metabolites production
Plants have been used throughout the world for its medicinal powers since ancient time. The pharmacological properties of plants are based on their phytochemical components especially the secondary metabolites which are outstanding sources of value-added bioactive compounds. Secondary metabolites have complex chemical composition and are produced in response to various forms of stress to perform different physiological tasks in plants. They are used in pharmaceutical industries, cosmetics, dietary supplements, fragrances, dyes, etc. Use of these metabolites in industries has initiated a need to focus research on increasing the production by employing plant tissue culture (PTC) techniques and optimizing their large-scale production using bioreactors. PTC techniques being independent of climatic and geographical conditions will provide an incessant, sustainable, economical and viable production of secondary metabolites (Chandran et al. 2020). Different medicinal plants produce different phytoconstituents like alkaloids, flavonoids and pterocarpans. The in vitro micropropagation method can serve as a valuable method in order to produce number of secondary metabolites (Sharma et al. 2021).
Callus culture in secondary metabolite production
Plant growth regulators are one of the most important factors affecting cell growth, differentiation and metabolite formation. The appropriate concentration of the medium is one of the critical determinants in controlling callus growth and metabolite production. To produce secondary metabolites from medicinal plants, it is important to establish the optimal culture conditions (chemical and physical environments) for the particular plant species. Oxidative stress plays an important role in the production of secondary metabolites in plants. Phenolic compounds are considered to be secondary metabolites that are synthesized in plants through the phenylpropanoid pathway and function as a defense mechanism that reacts to various biotic and abiotic stress conditions. The exposure of plants to unfavorable conditions leads to the generation of reactive oxygen species (ROS). The well-organized way to enhance the secondary metabolite production in callus suspension cultures was deeply analyzed in Bletilla striata (Pan et al. 2020).
Medicinal plants are important source of SMs which are directly or indirectly used by various pharmaceutical industries due to which the supply of these SMs is limited. The limited availability of biologically active, commercially valuable and medicinally important plant SMs can be overcome by using metabolic engineering and biotechnological processes. Advances in these techniques, particularly plant cell, tissue and organ cultures, provide valuable method for the production of medicinally important SMs. In cell cultures, suspension culture and elicitation are important strategy to enhance the SMs production. In various plants, these methods proved to be valuable tool for the production of SMs. Hairy root culture is an important method for enhancement of SMs production by using Agrobacterium rhizogenes. In some cases, hairy root cultures are considered most reliable than cell cultures for the commercial production of SMs. Shoot cultures and hairy root culture are useful because they provide a constant and reliable basis for the SMs production. The other advantage of these techniques is that they are independent of various geographical, seasonal and environmental conditions. Biotransformation helps in discovering the new compounds for pharmacological activity and for other compounds modifying their chemical structures allowing them to show pharmacological activities. In addition, biotransformation also provides synthesis of new compound in suspension culture of medicinally important plants. The use of genetic engineering, regulation of biosynthetic pathways of desired plant metabolites offers the production of commercially valuable secondary metabolites. Many molecular biology techniques, which are used in tissue cultures, induced the SM production by effecting the expression and regulation of biosynthetic pathways. In recent years, most of the in vitro strategies are used due to knowledge and regulation of SM pathway in commercially valuable plants. In future, these techniques will provide a valuable method to sustained feasibility of medicinal plant as renewable source of medicinally important compounds and these methods will provide successful production of desired, important, valuable and also unknown compounds.
Availability of data and materials
Ackermann C (1977) Pflanzen aus Agrobacterium rhizogenes-Tumoren an Nicotiana tabacum. Plant Sci Lett 8:23–30
Ahmad Z, Shahzad A, Sharma S (2019) Chitosan versus yeast extract driven elicitation for enhanced production of fragrant compound 2-hydroxy-4-methoxybenzaldehyde (2H4MB) in root tuber derived callus of Decalepis salicifolia (Bedd. ex Hook. f.) Venter. Plant Cell Tissue Organ Cult 136:29–40
Alvarez MA, Rodrfguez Talou J, Paniego NB, Giulietti AM (1994) Solasodine production in transformed organ cultures (roots and shoots) of Solanum eleagnifolium Cav. Biotechnol Lett 16:393–396
Balandrin MF, Klocke JA (1988) Medicinal, aromatic, and industrial materials from plants. In: Medicinal and aromatic plants. Springer, Berlin, pp 3–36
Baque MA, Moh SH, Lee EJ, Zhong JJ, Paek KY (2012) Production of biomass and useful compounds from adventitious roots of high-value added medicinal plants using bioreactor. Biotechnol Adv 30:1255–1267
Barrales HJ, Reyes CR, Garcia IV, Valdez LGL, De Jesus AG, Ruiz JAC, Montoya JM (2019) Alkaloids of Pharmacological Importance in Catharanthus roseus. In: Alkaloids-their importance in nature and human life. Intech Open
Begum F (2011) Augmented production of vincristine in induced tetraploids of Agrobacterium transformed shooty teratomas of Catharanthus roseus. Med Plants Int J Phytomed Relat Ind 3:59–64
Begum F, Nageswara Rao SSS, Rao K, Prameela Devi Y, Giri A, Giri CC (2009) Increased vincristine production from Agrobacterium tumefaciens C58 induced shooty teratomas of Catharanthus roseus G. Don. Nat Prod Res 23:973–981
Benedito AV, Modolo VL. Introduction to Metabolic Genetic Engineering for the Production of Valuable Secondary Metabolites in in vivo and in vitro Plant Systems. Recent Patents Biotechnol. 2014;8(1):61–75.
Bhadra R, Morgan JA, Shanks JV (1998) Transient studies of light-adapted cultures of hairy roots of Catharanthus roseus: growth and indole alkaloid accumulation. Biotechnol Bioeng 60:670–678
Binns AN, Thomashow MF (1988) Cell biology of Agrobacterium infection and transformation of plants. Annu Rev Microbiol 42:575–606
Bourgaud F, Gravot A, Milesi S, Gontier E (2001) Production of plant secondary metabolites: a historical perspective. Plant Sci 161:839–851
Chandran H, Meena M, Barupal T, Sharma K. Plant tissue culture as a perpetual source for production of industrially important bioactive compounds. Biotechnol Rep. 2020;26:00450.
Chattopadhyay S, Farkya S, Srivastava AK, Bisaria VS (2002) Bioprocess considerations for production of secondary metabolites by plant cell suspension cultures. Biotechnol Bioprocess Eng 7:138
Chodisetti B, Rao K, Gandi S, Giri A (2013) Improved gymnemic acid production in the suspension cultures of Gymnema sylvestre through biotic elicitation. Plant Biotechnol Rep 7:519–525
Christie PJ (1997) Agrobacterium tumefaciens T-complex transport apparatus: a paradigm for a new family of multifunctional transporters in eubacteria. J Bacteriol 179:3085
Constabel F (1990) Medicinal plant biotechnology1. Planta Med 56:421–425
DiCosmo F, Misawa M (1995) Plant cell and tissue culture: alternatives for metabolite production. Biotechnol Adv 13:425–453
DiCosmo F, Misawa M (1985) Eliciting secondary metabolism in plant cell cultures. Trends Biotechnol 3:318–322
Doran PM (2002) Properties and applications of hairy root cultures. In: Plant biotechnology and transgenic plants. CRC Press, pp 134–151
Ellis BE (1988) Natural products from plant tissue culture. Nat Prod Rep 5:581–612
Fujita Y, Tabata M (1986) Secondary metabolites from plant cells: pharmaceutical applications and progress in commercial production. Plant Biol (USA)
Funk C, Brodelius P (1990) Influence of growth regulators and an elicitor on phenylpropanoid metabolism in suspension cultures of Vanilla planifolia. Phytochemistry 29:845–848
Gelvin SB (2000) Agrobacterium and plant genes involved in T-DNA transfer and integration. Annu Rev Plant Biol 51:223–256
Ghorpade RP, Chopra A, Nikam TD (2011) Influence of biotic and abiotic elicitors on four major isomers of boswellic acid in callus culture of “Boswellia serrata” Roxb. Plant Omics 4:169
Giri A, Narasu ML (2000) Transgenic hairy roots: recent trends and applications. Biotechnol Adv 18:1–22
Giri A, Banerjee S, Ahuja PS, Giri CC (1997) Production of hairy roots in Aconitum heterophyllum wall using Agrobacterium rhizogenes. In Vitro Cell Dev Biol Plant 33:280–284
Giri A, Dhingra V, Giri CC (2001) Biotransformations using plant cells, organ cultures and enzyme systems: current trends and future prospects. Biotechnol Adv 19:175–199
Hamburger M, Hostettmann K (1991) Bioactivity in plants: the link between phytochemistry and medicine. Phytochemistry 30:3864–3874
Hamill JD, Rhodes MJC (1993) Manipulating secondary metabolism in culture. In: Biosynthesis and manipulation of plant products. Springer, Dordrecht, pp 178–209
Hao X, Pu Z, Cao G, You D, Zhou Y, Deng C, Shi M, Nile SH, Wang Y, Zhou W, Kai G (2020) Tanshinone and salvianolic acid biosynthesis are regulated by SmMYB98 in Salvia miltiorrhiza hairy roots. J Adv Res 23:1–12
Hema C, Mukesh M, Tansukh B, Kanika S (2020) Plant tissue culture as a perpetual source for production of industrially important bioactive compounds. Biotechnol Rep 26:1–10
Hu ZB, Du M (2006) Hairy roots and its application in plant genetic engineering. J Integr Plant Biol 48:121–127
Hussain MS, Fareed S, Saba Ansari M, Rahman A, Ahmad IZ, Saeed M (2012) Current approaches toward production of secondary plant metabolites. J Pharm Bioallied Sci 4:10
Ishihara K, Hamada H, Hirata T, Nakajima N (2003) Biotransformation using plant cultured cells. J Mol Catal B Enzym 23:145–170
Karuppusamy S (2009) A review on trends in production of secondary metabolites from higher plants by in vitro tissue, organ and cell cultures. J Med Plants Res 3:1222–1239
Khan T, Khan MA, Karam K, Ullah N, Mashwani ZU, Nadhman A (2021) Plant in vitro culture technologies; a promise into factories of secondary metabolites against COVID-19. Front Plant Sci 15:1–21
Kim Y, Wyslouzil BE, Weathers PJ (2002a) Secondary metabolism of hairy root cultures in bioreactors. In Vitro Cell Dev Biol Plant 38:1–10
Kim YJ, Weathers PJ, Wyslouzil BE (2002b) Growth of Artemisia annua hairy roots in liquid-and gas-phase reactors. Biotechnol Bioeng 80:454–464
Kreis W, Reinhard E (1989) The production of secondary metabolites by plant cells cultivated in bioreactors1. Planta Med 55:409–416
Krolicka A, Szpitter A, Stawujak K, Baranski R, Gwizdek-Wisniewska A, Skrzypczak A, Lojkowska E (2010) Teratomas of Drosera capensis var. alba as a source of naphthoquinone: ramentaceone. Plant Cell Tissue Organ Cult 103:285–292
Kumar V, Jones B, Davey MR (1991) Transformation by Agrobacterium rhizogenes of transgenic shoots of the wild soyabean. Glycine Argyria Plant Cell Rep 10:135–138
Longo MA, Sanroman MA (2006) Production of food aroma compounds: microbial and enzymatic methodologies. Food Technol Biotechnol 44:335–353
Yeoman MM (1987) Techniques, characteristics, properties and commercial potential of immobilized plant cells. Cell Cult Somat Cell Genet Plants 4:197–215
Massot B, Milesi S, Gontier E, Bourgaud F, Guckert A (2000) Optimized culture conditions for the production of furanocoumarins by micropropagated shoots of Ruta graveolens. Plant Cell Tissue Organ Cult 62:11–19
Moyano E, Palazon J, Bonfill M (2007) Biotransformation of hyoscyamine into scopolamine in transgenic tobacco cell cultures. J Plant Physiol 164:521–524
Mukundan U, Dawda HG, Ratnaparkhi S (1997) Hairy root culture and secondary metabolite production: Agrobacterium rhizogenes mediated transformed root cultures (No. 2). Agro Botanica
Mulabagal V, Tsay HS (2004) Plant cell cultures-an alternative and efficient source for the production of biologically important secondary metabolites. Int J Appl Sci Eng 2:29–48
Murthy HN, Hahn EJ, Paek KY (2008) Adventitious root and secondary metabolism. Chin J Biotechnol 24:711–716
Nguyen C, Bourgaud F, Forlot P, Guckert A (1992) Establishment of hairy root cultures of Psoralea species. Plant Cell Rep 11:424–427
Nilsson O, Olsson O (1997) Getting to the root: the role of the Agrobacterium rhizogenes rol genes in the formation of hairy roots. Physiol Plant 100:463–473
Ooms G, Twell D, Bossen ME, Hoge JHC, Burrell MM (1986) Developmental regulation of RI T L-DNA gene expression in roots, shoots and tubers of transformed potato (Solanum tuberosum cv. Desirée). Plant Mol Biol 6:321–330
Pan Y, Li L, Xiao S, Chen Z, Sarsaiya S, Zhang S, Guan YS, Liu H, Xu D (2020) Callus growth kinetics and accumulation of secondary metabolites of Bletilla striata Rchb.f. using a callus suspension culture. PLoS ONE 10:1–14
Petit A, David C, Dahl GA, Ellis JG, Guyon P, Casse-Delbart F, Tempe AJ (1983) Further extension of the opine concept: plasmids in Agrobacterium rhizogenes cooperate for opine degradation. Mol Gen Genet 190:204–214
Putalun W, Udomsin O, Yusakul G, Juengwatanatrakul T, Sakamoto S, Tanaka H (2010) Enhanced plumbagin production from in vitro cultures of Drosera burmanii using elicitation. Biotech Lett 32:721–724
Radman R, Saez T, Bucke C, Keshavarz T (2003) Elicitation of plants and microbial cell systems. Biotechnol Appl Biochem 37:91–102
Rao SR, Ravishankar GA (2002) Plant cell cultures: chemical factories of secondary metabolites. Biotechnol Adv 20:101–153
Rodney C, Toni M, Kutchan N, Lewis G (2000) Natural products. In: Buchanan B, Gruissem Jones WR (eds) Biochemistry and molecular biology of plants, pp 1253–348
Rudrappa T, Neelwarne B, Kumar V, Lakshmanan V, Venkataramareddy SR, Aswathanarayana RG (2005) Peroxidase production from hairy root cultures of red beet (Beta vulgaris). Electron J Biotechnol 8:66–78
Saito K, Murakoshi I, Inze D, Montagu M (1985) Biotransformation of nicotine alkaloids by tobacco shooty teratomas induced by a Ti plasmid mutant. Plant Cell Rep 7:607–610
Saito K, Yamazaki M, Kawaguchi A, Murakoshi I (1991) Metabolism of solanaceous alkaloids in transgenic plant teratomas integrated with genetically engineered genes. Tetrahedron 47:5955–5968
Siebert M, Sommer S, Li SM, Wang XZ, Severin K, Heide L (1996) Genetic engineering of plant secondary metabolism. Accumulation of 4-hydroxybenzoate glucosides as a result of the expression of the bacterial ubiC gene in tobacco. Plant Physiol 112:811–819
Sevon N, Oksman-Caldentey KM (2002) Agrobacterium rhizogenes-mediated transformation: root cultures as a source of alkaloids. Planta Med 68:859–868
Shanks JV, Morgan J (1999) Plant ‘hairy root’ culture. Curr Opin Biotechnol 10:151–155
Sharma S, Sharma S, Sharma V (2021) Exploring In-vitro micropropagation and secondary metabolite production in Desmodium gangeticum (L.) D.C. I.K. Press 22:231–240
Shen WH, Petit A, Guern J, Tempe J (1988) Hairy roots are more sensitive to auxin than normal roots. Proc Natl Acad Sci 85:3417–3421
Shimoda K, Kondo Y, Nishida T (2006) Biotransformation of thymol, carvacrol, and eugenol by cultured cells of Eucalyptus perriniana. Phytochemistry 67:2256–2261
Shimoda K, Kwon S, Utsuki A (2007) Glycosylation of capsaicin and 8-nordihydrocapsaicin by cultured cells of Catharanthus roseus. Phytochemistry 68:1391–1396
Simeo Y, Sinisterra JV (2009) Biotransformation of terpenoids: a green alternative for producing molecules with pharmacological activity. Mini-Rev Org Chem 6:128–134
Singh G (1990) Elicitation—manipulating and enhancing secondary metabolite production. In: Plant cell and tissue culture for the production of food ingredients, pp 101–111
Smetanska I (2008) Production of secondary metabolites using plant cell cultures. In: Food biotechnology, pp 187–228
Spencer A, Hamill JD, Rhodes MJ (1990) Production of terpenes by differentiated shoot cultures of Mentha citrata transformed with Agrobacterium tumefaciens T37. Plant Cell Rep 8:601–604
Spencer A, Hamill JD, Rhodes MJ (1993) In vitro biosynthesis of monoterpenes by Agrobacterium transformed shoot cultures of two Mentha species. Phytochemistry 32:911–919
Stewart FC, Rolfs FM, Hall FH (1900) A fruit-disease survey of western New York in 1900 (No. 191). New York Agricultural Experiment Station
Subroto MA, Kwok KH, Hamill JD, Doran PM (1996) Coculture of genetically transformed roots and shoots for synthesis, translocation, and biotransformation of secondary metabolites. Biotechnol Bioeng 49:481–494
Sudha G, Ravishankar GA (2003) Elicitation of anthocyanin production in callus cultures of Daucus carota and involvement of calcium channel modulators. Curr Sci 84:775–779
Tepe B, Sokmen A (2007) Production and optimisation of rosmarinic acid by Satureja hortensis L. callus cultures. Nat Prod Res 21:1133–1144
Tzfira T, Citovsky V (2000) From host recognition to T-DNA integration: the function of bacterial and plant genes in the Agrobacterium–plant cell interaction. Mol Plant Pathol 1:201–212
Tzfira T, Citovsky V (2002) Partners-in-infection: host proteins involved in the transformation of plant cells by Agrobacterium. Trends Cell Biol 12:121–129
Tzfira T, Rhee Y, Chen MH, Kunik T, Citovsky V (2000) Nucleic acid transport in plant-microbe interactions: the molecules that walk through the walls. Annu Rev Microbiol 54:187–219
Vagner AB, Luzia VM (2014) Introduction to metabolic genetic engineering for the production of valuable secondary metabolites in in vivo and in vitro plant systems. Recent Patents Biotechnol 8:61–75
Vanisree M, Chen YL, Shu-Fung L, Satish MN, Chien YL, HsinSheng T (2004) Studies on the production of some important secondary metabolites from medicinal plants by plant tissue cultures. Bott Bull Acad Sin 45:1–22
Vasil IK, Constabel F, Schell J (1984) Cell culture and somatic cell genetics of plants (1). Academic Press, New York
Veersham C (2004) Medicinal plant biotechnology. CBS Pub, New Delhi, pp 377–419
Verpoorte R, Contin A, Memelink J (2002) Biotechnology for the production of plant secondary metabolites. Phytochem Rev 1:13–25
Wallaart TE, Pras N, Quax WJ (1999) Isolation and identification of dihydroartemisinic acid hydro peroxide from Artemisia annua: a novel biosynthetic precursor of artemisinin. J Nat Prod 62:1160–1162
Ye M, Guo D (2005) Substrate specificity for the 12β-hydroxylation of bufadienolides by Alternaria alternata. J Biotechnol 117:253–262
Ye M, Han J, Guo H, Guo D (2002) Structural determination and complete NMR spectral assignments of a new bufadienolide glycoside. Magn Reson Chem 40:786–788
Ye M, Han J, Tu G, An D, Guo D (2005) Microbial hydroxylation of bufalin by Cunninghamella blakesleeana and Mucor spinosus. J Nat Prod 68:626–628
Ye M, Ning L, Zhan J (2003) Biotransformation of cinobufagin by cell suspension cultures of Catharanthus roseus and Platycodon grandiflorum. J Mol Catal B Enzym 22:89–95
Ye M, Qu G, Guo H, Guo D (2004) Specific 12β-hydroxylation of cinobufagin by filamentous fungi. Appl Environ Microbiol 70:3521–3527
Zhang J, Sun Y, Liu JH, Yu BY, Xu Q (2007) Microbial transformation of three bufadienolides by Nocardia sp. and some insight for the cytotoxic structure–activity relationship (SAR). Bioorg Med Chem Lett 17:6062–6065
Zhao J, Guan SH, Chen XB, Wang W, Ye M, Guo DA (2007) Two new compounds derived from bufalin. Chin Chem Lett 18:1316–1318
Zhou Y, Hirotani M, Yoshikava T, Furuya T (1997) Flavonoids and phenylethanoids from hairy root cultures of Scutellaria baicalensis. Phytochemistry 44:83–87
Zupan J, Muth TR, Draper O, Zambryski P (2000) The transfer of DNA from Agrobacterium tumefaciens into plants: a feast of fundamental insights. Plant J 23:11–28
Ethics approval and consent to participate
Consent for publication
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
About this article
Cite this article
Fazili, M.A., Bashir, I., Ahmad, M. et al. In vitro strategies for the enhancement of secondary metabolite production in plants: a review. Bull Natl Res Cent 46, 35 (2022). https://doi.org/10.1186/s42269-022-00717-z