Keywords

1 Introduction

Plants are able to produce a wide range of primary and secondary metabolites (Yazaki et al. 2008; Sharma et al. 2013). However, the large-scale production of these valuable compounds has been limited by low growth rates, climate dependency, restricted cultivation areas, plant diseases, pests, overharvesting, and intense labor requirement (Sharma et al. 2013; Meena et al. 2013a; Bahadur et al. 2014; Maurya et al. 2014; Jat et al. 2015; Kumar et al. 2016b; Rates 2001). Moreover, the chemical synthesis of plant-derived metabolites is not a feasible choice due to their complex structures and their specific stereochemical requirements (Sharma et al. 2013; Namdeo 2007). All these issues emphasize the need for developing new methods and protocols for the industrial-level fabrication of plant-derived metabolites.

Suspension culture of plant cells has been considered as another promising source for biosynthesis of valuable secondary metabolites (Sharma et al. 2013). More than 25% of the available pharmaceuticals are either based on originally found compounds in plants or are extracted from them (Giri and Narasu 2000). Production of secondary metabolites by using suspended plant cell culture is usually a challenging task as these compounds produce at distinct developmental stages. Therefore, in vitro studies of differentiated and organized tissues (mainly the roots) have been developed and were reported to be a more predictable approach as compared to cell suspension cultures (Sharma et al. 2013; Kumar et al. 2015; Ahmad et al. 2016; Meena et al. 2016a; Parewa et al. 2014).

The plant roots are suitable for large-scale production since they are the key point for synthesis and/or storage of certain chemicals. The biotechnological fabrication of wide range of valuable secondary metabolites by using plant cultures can be seen as an alternative to the extraction of whole plant material (Namdeo 2007). Several strategies have been investigated in order to further enhance the production of secondary metabolites from medicinal plants. Some of these include high yielding cell line screening, media modification, elicitation, precursor feeding, large-scale cultivation system, plant cell immobilization, hairy root culture, biotransformation, and others (Rao and Ravishankar 2002; Vanisree et al. 2004).

Recently, hairy root (HR) culture has been developed in order to inhibit the use of large volumes of plants that are needed to be purified. Totipotency is among the major characteristics of plant cells; therefore, HRs could successfully produce primary and secondary metabolites similar to intact roots (Giri and Narasu 2000; Qaderi et al. 2016). HR culture is a tool that makes use of soil bacterium Agrobacterium rhizogenes ability to transfer genes to the genome of the host plant (Sharma et al. 2013; Thwe et al. 2016). This technique was developed as the innovative path for bulky production of secondary metabolite and phytochemicals which allows developing large amount of roots and secondary metabolites in short time for continuous supply of improved value products (Korde et al. 2016). These HRs have also been used for root physiology and biosynthetic pathway (Giri and Narasu 2000), regeneration of whole plants with desirable phenotypes, and phytoremediation of toxic substances and reactive dyes (Talano et al. 2012; Prakash and Verma 2016; Meena et al. 2015a, 2016b; Priyadharsini and Muthukumar 2016; Kumar et al. 2017). Finally, HR cultures include other aspects such as molecular metabolic engineering, bioreactor design, and optimization (Ono and Tian 2011). In this chapter, we present advances in work on HR cultures related to the detoxification of pollutants, production of valuable metabolites, and fabrication in large-scale intensified bioreactors. A suggestion to overcome current challenges and emerging trends for future progression of research has also been provided.

2 Definition and Basic Features of HRs

HR production is carried out through the plant tissue culture technique in order to study the plant metabolic processes or to manufacture precious secondary metabolites with the use of plant genetic engineering. HR culture is also called as transformed root culture from gram-negative soil bacterium A. rhizogenes that contains root-inducing plasmids (Ri plasmids) (Korde et al. 2016; Pistelli et al. 2010). It infects roots of dicot, and some monocot plants cause them to produce the opines which is a type of unusual amino acids (octopine, agropine, nopaline, mannopine, and cucumopine). Such opines are used by the bacterium as a carbon, nitrogen, and energy source (Ferdosi and Kashefi 2014).

The morphology of HRs is significantly different from the normal roots as they are much more branched and have much lateral meristematic growth, which will lead to higher biomass. The abnormal roots however are easier to grow in artificial media without hormone, and they are neoplastic in nature, with hazy growth. Fabricated HRs by infection of A. rhizogenes have a high growth rate as well as genetic and biochemical makeup (Korde et al. 2016).

However, new techniques are developed in order to make HRs by the use of new plant species (Georgiev et al. 2011). HRs have numerous advantages such as indefinite and fast in vitro growth even in the absence of phytohormones as well as high genotype and phenotype stability (Ono and Tian 2011). HR culture is among the main cultures that are used to investigate metabolic processes of plants, secondary metabolites production, recombinant proteins, plant genetic engineering, phytoremediation, artificial seed production, biofortification, and biopharmaceuticals. Applications of these efficient technologies also include several aspects as metabolic engineering, bioreactor design, and process optimization (Raghavendra et al. 2016; Zahedi 2016; Meena et al. 2015b; Rawat et al. 2016; Kumar et al. 2016a).

3 Mechanism of HR Cultures

The interaction between plants and A. rhizogenes in HR establishment involves a complex series of events. A. rhizogenes is responsible for a neoplastic outgrowth of fine roots at the infection site, and infected plants show reduced vitality. These symptoms came to be known as the hairy root disease. Roots arising at the site of infection can be cultured aseptically, and the resulted transformed root clones may be subcultured indefinitely on basal medium, growing at rates several times quicker than normal roots (Flores and Filner 2012). Agrobacterium recognizes various phenolic compounds that have been produced from wounded plant cells, namely, acetosyringone and α-hydroxy acetosyringone.

After microbial colonization, consequently their attachment to plant cells T-DNA will be inserted (Sharma et al. 2013). This T-DNA is a transferable DNA from bacterium to plant cell. The T-DNA has a set of genes that are capable to encode enzymes required for cytokinin biosynthesis, phytohormone auxin control (iaaM, iaaH, ipt), and synthesis of sugar and amino acids (unusual amino acids). These segments have eukaryotic regulatory sequences and approximately 10–30 kbp in size and encode for the Ri conjugation, catabolism, opine synthesis, and integration of the T-DNA itself (Pistelli et al. 2010). Genes of T-DNA fragment facilitate the formation of neoplastic crown gall tumor and HR tissues followed by the synthesis of opines (Sharma et al. 2013; Yasin et al. 2016; Meena et al. 2016c; Jaiswal et al. 2016; Jha and Subramanian 2016). Depending on the bacterial strain, these metabolites are used as a carbon and nitrogen source for the bacteria. A. rhizogenes strains were categorized into two main classes, namely, Agropine-type and Mann opine-type strains. Among these, agropines are the most often used strains due to their strongest virulence (Sharma et al. 2013). The virulent strains of A. rhizogenes contain the Ri plasmids with different gene sequences (Fig. 7.1). Plasmids can be divided in strains producing mannopine and cucumopine with single DNA and strains producing octopine and agropine with two T-DNAs. The two T-DNAs are classified in the TR-DNA (right DNA) and the TL-DNA (left DNA). The root-inducing genes (rol A, rol B, rol C, rol D) are found in the center of TL-DNA of the agropine-producing strains. Parts of the TR-DNA are genes for the biosynthesis of auxins and the synthesis of mannopine and agropine. After the transfer of the TL-DNA and TR-DNA, they are integrated in the genome of the plant cell. The TL-DNA is vital for the hairy root induction (Chandra 2012).

Fig. 7.1
figure 1

Schematic overview of a Ri plasmid of A. rhizogenes

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4 Establishment of HR Cultures

Successful HR culture system requires several essential check marks, namely, selection of best A. rhizogenes strain, appropriate explants and antibiotic, and a suitable culture medium (Sharma et al. 2013). Strains of A. rhizogenes are widely varying in their transforming ability. HRs that are fabricated by using different types of bacterial strains show significantly different morphologies. These observed virulence and morphology differences could be justified by the different strain plasmid harbored (Saha et al. 2016a; Yadav and Sidhu 2016; Nguyen et al. 1992; Meena et al. 2015f). Most plant materials like hypocotyl, stem, cotyledon, leaf, tuber, or storage root may be applied to make HRs (Królicka et al. 2001; Sevón and Oksman-Caldentey 2002; Giri et al. 2001). In order to induce HRs, explants should be infected with strains of A. rhizogenes either by cocultivation or direct inoculation (Giri et al. 2001; Ur Rahman et al. 2004). Subsequently, roots are subculture using a medium such as MS or B5 (Fig. 7.2) (Sevón and Oksman-Caldentey 2002; Le Flem-Bonhomme et al. 2004; Palazón et al. 2003a, b).

Fig. 7.2
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An overview of the HR culture establishment

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5 Application of HR Cultures

The HR culture shaves diverse and abundant applications (Fig. 7.3). They traditionally have been used to investigate root physiology in conjunction with biosynthetic pathway elucidation (Ibanez et al. 2016). Nowadays, HR culture technique is now being used for the fabrication of bioactive compounds, secondary metabolites, and phytochemicals. HRs are popular for regeneration of whole plants with desirable phenotypes by infection of ornamental plants with A. rhizogenes (Meena et al. 2016d; Saha et al. 2016b; Verma et al. 2015b; Bahadur et al. 2016b; Das and Pradhan 2016; Dominguez-Nunez et al. 2016; Dotaniya et al. 2016).

Fig. 7.3
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The systematic applications of HR cultures

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Moreover, HR cultures have been used for phytoremediation of toxic substances and reactive dye. Presently, several high-value bioactives are fabricated by using HRs from various plant sources which have application in pharmaceutical and cosmetic products (Ono and Tian 2011). Furthermore, recombinant protein production using this system was found to be a sustainable method for producing cytokines as well as protein therapeutics (Talano et al. 2012).

5.1 Application of HR Cultures for Secondary Metabolites Production

Plants are known as chemical factories since they pose the ability to fabricate important phytochemical. However, the major issue is that their growth is dependent to the outside environmental stress. HR cultures are promising source for phytochemical due to sizable biomass production and biosynthetic capacity.

Furthermore, HR cultures frequently accumulate phytochemical at much higher levels than cell or callus cultures (McCoy and O’Connor 2008; Ono and Tian 2011). Secondary metabolites as summarized in Table 7.1 are naturally more complex as compared to primary metabolites. These compounds have been categorized into terpenoids, phenolics, and alkaloids (Chinou 2008).

Table 7.1 Classification of secondary metabolites (Rao and Ravishankar 2002)
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Several HR cultures have attracted significant amount of attention due to their potential in production of valuable phytochemical including Artemisia annua, Catharanthus roseus, Arachis hypogaea, and Camptotheca acuminata/Ophiorrhiza pumila (McCoy and O’Connor 2008). List of secondary metabolites produced by wild HR cultures is summarized in Table 7.2. Although, non-transgenic HR cultures continue to serve as a good source for phytochemicals and secondary metabolites (Ono and Tian 2011).

Table 7.2 List of the secondary metabolites that are produced by wild HR cultures
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However, application of metabolic engineering methods requires acritical understanding about the regulation of secondary metabolite pathways and the metabolic phenotype of the HR culture to regulate the dynamic distribution of metabolites between different biochemical pathways (Kruger and Ratcliffe 2009; Talano et al. 2012). Table 7.3 tabulated the common secondary metabolites that are produced by transgenic HRs.

Table 7.3 List of secondary metabolites produced through transgenic HR cultures (Talano et al. 2012)
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5.2 Application of HR Cultures in Phytoremediation

Environmental remediation is a method that deals with the removal of toxins in order to support the environment (Macek et al. 2008). Remediation processes can be expensive; therefore, efficient and inexpensive technologies are still developing to address the needs in the field. Bioremediation includes the environmental treatment with the use of microorganisms and plants (Novakova et al. 2007; Najmanova et al. 2007). In case of using plants, this is so-called phytoremediation (Garbisu and Alkorta 2001; Korde et al. 2016). The use of plants to absorb and accumulate organic and inorganic pollutants or to transform toxic molecules to harmless once has attracted attention (Eapen et al. 2007; Doty 2008; Macek et al. 2008; Ibanez et al. 2016).

Phytoremediation process usually occurs through several complex interactions between the key involved sources (Krystofova et al. 2009; Guillon et al. 2006). Roots are normally the main contact point between contaminants and the plant tissues they are the key point of assessment of the phytoremediation potential (Verma et al. 2014; Meena et al. 2014a, 2015e; Teotia et al. 2016).

However, in this sense HR culture has been found suitable in order to study the xenobiotic detoxification without the soil matrix or microbes interaction (Talano et al. 2012). Figure 7.4 summarizes how a plant deposits the toxin efficiently. Plants are able to chemically modify toxic substances through their direct metabolism (Krystofova et al. 2009). Greater genotypic and phenotypic stability are the important advantages of HR cultures which provide a more promising system over time for phytoremediation (Doran 2009). Additionally, the organized nature of HR cultures offers an added advantage that makes them more useful for cultivation in bioreactors at large scale (Angelini et al. 2011; Bahadur et al. 2017; Verma et al. 2017b; Kumar et al. 2017b). HRs that have been functionalized by genetic engineering are expected to become a new solution for environmental treatment in near future (Guillon et al. 2006).

Fig. 7.4
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Types of phytoremediation. Plant metabolizes the pollutant via organic and inorganic phytoremediation or rhizospheric metabolism. Phytoremediation includes several approaches, namely, phytostabilization, phytoextraction, phytodegradation, and phytostimulation

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HRs produced by hyper-accumulators are capable to uptake nickel, uranium, or cadmium from polluted environment (Boominathan and Doran 2003; Boominathan et al. 2004; Eapen et al. 2003; Agostini et al. 2003; Suresh et al. 2005; Gujarathi et al. 2005). Phytoremediation of several environmental pollutants by wild-type and transgenic HR cultures is shown in Table 7.4.

Table 7.4 Phytoremediation of several environmental pollutants by wild-type and transgenic HR cultures
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6 Recent Advances in HR Cultures Scale-Up

Root tissues are not identical to microbial cultures in many ways. Therefore, bioreactors for HR cultures are more challenging to be controlled, operated, and scaled up. Development of innovative bioreactors and process intensification will allow to optimize cell growth and large-scale production (Sharma et al. 2016; Meena et al. 2013c, 2016e; Verma et al. 2015a; Bahadur et al. 2016a; Masood and Bano 2016).

6.1 Development of Groundbreaking Bioreactors in HR Cultures

Design and optimization of bioreactors have been the great advance in HRs for industrial-scale production of metabolites (Huang and McDonald 2012). Production of HRs in bioreactors helps to have a better control on operating conditions and consequently optimize the growth and biosynthesis of the secondary metabolite (Eibl and Eibl 2008). Bioreactor optimization for fabrication of HRs is of critical importance for scale-up strategies.

HR bioreactors can be in general divided into gas or liquid phase. In liquid-phase bioreactors, roots are always placed in the medium; as a result they are called submerged reactors. On the other hand, in gas-phase reactors, the roots are almost exposed to air or another gas mixture (Kim et al. 2002a, b; Stiles and Liu 2013). The design of the reactor also depends on the product location, which is either intracellular or extracellular (Meena et al. 2013b, 2015d; Shrivastava et al. 2016; Singh et al. 2015).

HR cultivation is usually associated with clumps formation that are naturally composed of primary roots and their bridged lateral roots. It is very difficult to find appropriate bioreactor for HR cultures because the rheological properties of HR cultures vary from one species to another and even within clones of a single species. Several bioreactor designs have been reported for HRs (Mishra and Ranjan 2008). Schematic diagrams of promising bioreactor types that have been successfully tested are depicted in Fig. 7.5.

Fig. 7.5
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Schematic diagrams of the bioreactors used for HR culture

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6.1.1 Liquid-Phase Bioreactors

In liquid-phase reactors, the culture space is filled up with liquid medium, and several techniques are used to provide the required aeration to the media. Since the roots are submerged, therefore, mixing and mass transfer are the main issues in scaling up the process (Eibland Eibl 2008; Curtis 2000; Nath et al. 2017; Sarkar et al. 2017; Verma et al. 2017a).

Design considerations comprise of mechanisms to provide adequate nutrient to the roots. Mixing and aeration strategy represents the main design differences between the various types of liquid-phase bioreactors. The most commonly used liquid-phase bioreactors are, namely, pneumatic reactors and stirred tanks. Methods for immobilizing HRs include cages, meshes, and polyurethane foam (Eibl and Eibl 2008).

Past studies evaluated the use of stirred tank reactors (STR) for HRs cultivation. In order to supply required amount of oxygen, compressed air is always spared into the bioreactor from a placed device in the impeller region. STRs are normally not useful for HR cultures despite their wide range of application in biotechnology. This is mainly because of the callus formation and wound response which are usually a response to the impeller rotation shear stress (Taya et al. 1989; Mishra and Ranjan 2008). Pneumatic bioreactors are the ones that include both airlift and bubble column reactors.

Bubble column reactors (BCR) are among the simplest bioreactors that are easy to scale up. The use of bubbles instead of mechanical mixers minimizes the shear stress on the cultures (Choi et al. 2008; Huang and McDonald 2012). However, the major drawback with BCRs are the undefined flow pattern of the liquid (Choi et al. 2008) and the reduced growth performance (Kwok and Doran 1995). In the presence of high biomass, the bubbles may coalesce resulting in the reduction of gas-liquid interface area (Huang and McDonald 2012). BCRs are liquid-phase bioreactors in which the roots are submerged in the medium. Liquid mixing is obtained by the upflow of air bubbles generated from an air distributor situated at the bottom of the column. In contrast to BCRs, airlift reactors (ALRs) contain a draft tube (either internal or external) to avoid coalescing bubbles. ALRs distribute shear stress more evenly, reduce shear stress, consume little energy, and promote a cylindrical mixing of the medium (Stiles and Liu 2013).

The draft tube in ALRs prevents bubble coalescence by forcing the bubbles to move in one direction. It also distributes shear stresses equally throughout the reactor. As a result, cells are able to grow in a more stable physical environment than those growing under high shear, a condition causing cell damage and lower productivity in STRs. It has also been shown experimentally that shear stress rates generated in ALRs are lower than those generated in BCRs.

ALRs have been extensively used for HRs since the initiation of HR bioreactor studies for species including Panax ginseng (Yoshikawa and Furuya 1987), Armoracia rusticana (Taya et al. 1989), Trigonella foenum-graceum (Rodriguez-Mendiola et al. 1991), Lippia dulcis (Sauerwein et al. 1991), Lithospermum erythrorhizon (Shimomura et al. 1991), Ophiorrhiza pumila (Sudo et al. 2002), and Echinacea purpurea (Abbasi et al. 2009).

Conventional ALRs have been extensively used for scale-up cultures of HR lines; however, they are generally not appropriate for high-density cultures due to inadequate mixing and oxygen mass transfer (Choi et al. 2008). This phenomenon is mainly based on uneven distribution of root tissue at certain regions as well as excessive gas-phase channeling (Taya et al. 1989).

6.1.2 Gas-Phase Bioreactors

In gas-phase reactors, roots are exposed to a mixture of air or gas mixture. The liquid nutrient is usually sprayed onto the top of the root bed (Kim et al. 2002a, b). These reactors have been widely used in plant tissue and HR cultures due to their abundant oxygen supply (Stiles and Liu 2013; McKelvey et al. 1993; Katuri et al. 2011; Wyslouzil et al. 1997). However, gas-phase reactors yet require a matrix for anchoring the HRs. These can be mesh trays or mesh cylinders. In addition, these reactors are labor intensive as their requirement for uniform loading (Eibl and Eibl 2008; Choi et al. 2008; Srivastava and Srivastava 2007; Velazquez et al. 2016; Meena et al. 2014b, 2015c; Sindhu et al. 2016; Singh et al. 2016; Ramakrishnan and Curtis 2004).

Nutrient mist reactors (NMRs) are another one of gas-phase-type reactors. In these systems plant organ is usually dispersed in the air phase with the help of a mesh support. NMRs have definite advantages, such as easy operation, high dissolved oxygen tension present in the mist, lack of shear, and ease of scaling up. Whitney (1992) investigated the performance of different types of bioreactors for D. stramonium and Nicotiana tabacum cultivation. Authors stressed that the growth rate and yield of tobacco HRs were greater in NMRs than in STRs, TBRs, and ALRs. Recently, HRs of Stizolobium hassjoo (velvet bean) were cultivated in 3 and 9 l NMRs by Huang et al. (2004) to evaluate the oxygen uptake rate, effects of intermittent medium supply, and other growth-related parameters.

Several other comparative studies also have been evaluated the optimal bioreactor type for a particular species of HRs. The production of Artemisia annua HRs was compared both in bubble and mist bioreactors. Based on the results, authors suggested that the overall biomass was higher in the BCR (Kim et al. 2002a, b). However, the mist reactor usually accumulates lower amount of biomass as compared to the BCR. This behavior could be due to insufficient nutrient availability (Choi et al. 2008; Srivastava and Srivastava 2012a, b).

6.1.3 Novel Bioreactors

Hybrid reactor can be seen as a method to address this issue, a reactor which allows the roots to attach uniformly to the anchoring system (Stiles and Liu 2013). Disposable bioreactors could also be seen as another alternative to the traditional protocols. These reactors can significantly reduce the operation costs by eliminating the need for cleaning or sterilization though out the process (Eibl and Eibl 2006). Disposable wave bioreactor systems could also be another advancement in the bioreactor design area (Mishra and Ranjan 2008). These systems work on the basis of using wave for agitation purpose which in turn reduces the stress levels (Palazón et al. 2003a, b). Large-scale wave bioreactors having the capacities of up to 600 L are now commercially applicable (Mishra and Ranjan 2008; Eibl and Eibl 2006).

6.2 Process Intensification

Process intensification methods could also be utilized in plant and tissue culture works (Stiles and Liu 2013). The ability to exploit HR cultures as a source of bioactive chemicals depends on the development of a suitable bioreactor system where several physical and chemical parameters must be taken into consideration. Selection of highly productive cell lines, manipulation of nutrients, optimizing the culture environment, elicitation, metabolic engineering, in situ product removal, and ultrasound have been applied for process intensification in HR bioreactor cultures (Mishra and Ranjan 2008; Stiles and Liu 2013).

6.2.1 Optimization of Bioreactor Parameters

Development of an appropriate bioreactor depends on several physical and chemical parameters, such as optimum pH, sufficient substrate, controlled temperature, salts for nutrition, product and by-product removal, oxygen, inoculation size and density, and product recovery. The agro-bacterial concentration has an important role in the production of transformed roots (Mishra and Ranjan 2008). Dissolved oxygen is another important factor in the bioreactor microenvironment. HRs cultured in bioreactors have the affinity to form clumps which critically inhibits the oxygen transfer (Bordonaro and Curtis 2000). Nutrient availability is also a major point for scale-up, and minerals are an important regulatory factor for HR growth (Sivakumar et al. 2005). Furthermore, periodic measurement of nutrients concentration during periods in bioreactors would provide key information regarding metabolic production (Wilhelmson et al. 2006; Sivakumar et al. 2005).

Light also plays a key role for both growth and production of secondary metabolites. The stimulatory role of light on the production of secondary compounds has been demonstrated using plant species “Perilla frutescens and Artemisia annua” (Zhong et al. 1991; Wang et al. 2001; Abbasi et al. 2007; Taya et al. 1994; Jacob and Malpathak 2004).

6.2.2 Elicitation

Elicitation is the effective technique which is currently used for improving the production of secondary metabolites (Zhao et al. 2005; Baenas et al. 2014). Overall, based on origin, elicitors are classified biotic and abiotic. Basically, biotic elicitors are either physical factors or chemical factors such as ultraviolet light heavy metals and salts (Stiles and Liu 2013). Salts including AlCl3, AgNO3, CdCl2, CaCl2, CuCl2, CoCl2, KCl, HgCl2, MgSO4, VOSO4NiSO4, and Zn ions have been used to increase the secondary metabolite production in a variety of plant (Ramirez-Estrada et al. 2016; Li et al. 2006; Vasconsuelo and Boland 2007). Abiotic elicitors are usually cheaper than biotic; however, they are not as efficient for the cultivation of the target microorganism (Georgiev et al. 2007). Specificity of the elicitor, culture growth stage, treatment interval, the concentration, medium composition, and light are the main factors that affect the effectiveness of elicitation (Sharma et al. 2013).

6.2.3 Metabolic Engineering

Metabolic engineering of biosynthetic pathways has been established recently to enhance the fabrication of secondary metabolites. In this case some general issues have to be taken into account, namely, competing pathways, cofactors for the reaction, and rate-limiting enzymatic steps which are among the major metabolic engineering issues (Ludwig-Müller et al. 2014; Georgiev et al. 2010; Chandra and Chandra 2011).

7 Concluding Remark and Future Developments of HR Cultures

To date, significant progresses have been made in the genetic transformation and tissue culture in order to amplify the key pathways for the biosynthesis of targeted metabolites. Commercial production of HRs has attracted much attention recently as compared to the other plant cells. HR cultures are unique due to their much higher genetic and biosynthetic stability. However, exploration into inexpensive novel elicitors and bioreactors are required in order to warrant their industrial implementation. Further, generated knowledge from plant metabolic pathways and advancements in genetic engineering will help HRs to become a promising and sustainable fabrication system in the near future.