Keywords

9.1 Introduction

Hairy root formation is induced by Rhizobium rhizogenes (earlier known as Agrobacterium rhizogenes) bacteria (Gutierrez-Valdes et al. 2020), at the site of infection by integration of T-DNA of the root-inducing (Ri) plasmid into the host genome. The hairy roots usually grow faster than plant cell cultures and yield more biomass. These hairy roots show rapid growth in hormone-free medium with extensive lateral branching and ageotropic nature, making these roots to be used as an attractive system for producing secondary metabolites. Further, their high genetic stability ensures the uniform productivity for longer period, which also makes these hairy roots as preferred material for HRC-based in vitro production. Several phytochemicals of medicinal and commercial importance, i.e., pharmaceuticals, cosmetics, dye, pesticides, food colorant, and food additives, have been reported. These hairy roots have potential to be utilized for in vitro production of secondary metabolites. Though several in vitro culture methods such as callus culture, cell suspension culture, adventitious root culture, and organ culture are available for secondary metabolites (Singh et al. 2010; Silja and Satheeshkumar 2015; Purwianingsih et al. 2016; Shoja and Shishavani 2021), the genetically transformed hairy roots are very attractive and promising keeping in view their differentiated nature and genetic stability and high production and productivity of secondary metabolites. The hairy root culture (HRC)-based production systems for secondary metabolites were reported for shikonins, camptothecins, azadirachtin, paclitaxel, hyoscyamine, harpagide, ginsenosides, and scopolamine (Sim and Chang 1993; Satdive et al. 2007; Almagro et al. 2015; Zhang et al. 2017; Balasubramanian et al. 2018; Singh et al. 2020; Barba-Espin et al. 2020; Wawrosch and Zotchev 2021; Table 9.1). As the plant-based phytoceuticals are in great demand worldwide, as per the estimate of World Health Organization (WHO), up to 80% of people take traditional herbs as medicines (Khan et al. 2009).

Table 9.1 HRCs for secondary metabolite production
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HRCs are usually capable of producing the structurally identical compounds similar to the ones found in roots of the naturally occurring intact parent plant, in contrast to as frequently observed in callus or cell suspension cultures. In comparison to undifferentiated cell culture, higher and stable yield of secondary metabolites are obtained with differentiation hairy roots. HRCs usually surpass the spatial barrier of production or accumulation of metabolites and could be employed also in cases where secondary metabolites accumulate or produced in other aerial tissues (stem, leaf, fruit, flower, seeds). HRC is considered to be more biosynthetically efficient than their mother plants. The hairy roots are also used for the production of those metabolites, which are synthesized and accumulate in aerial part of plants only, for example, lawsone and artemisinin, so irrespective of their origin or site of production, the metabolites could be obtained in hairy roots (Patra and Srivastava 2016; Bakkali et al. 1997). High genetic stability, differentiated tissue type, and high productivity are the features of HRC leading it to be used as valuable alternative method for the production of plant secondary metabolites. High branching, high growth rate, and genetic stability of these roots also suit for commercial up-scaling even in bioreactors; hence, in recent years, focus has been on designing appropriate bioreactors suitable to culture the delicate and sensitive hairy roots (Rekha and Thiruvengadam 2017). But, the major bottlenecks for commercial exploitation of HRC are the developing protocol for scaling up production in bioreactors, as easy handling of interconnected hairy roots that are unevenly distributed in the vessel is difficult. In this chapter, we have discussed the HRC with latest information available on basic method of induction of hairy roots to its uses in production low volume and up-scaling in bioreactors for commercial viability of technology.

9.2 Molecular Mechanism of Hairy Root Development

Root loci (rol) genes present on T-DNA of the root-inducing (Ri) plasmid of R. rhizogenes are integrated into the host genome causing hairy root formation. The most studied agropine-type strains, R. rhizogenes strains, have two T-DNA regions designated as the TL-DNA and TR-DNA on their Ri plasmid (Nemoto et al. 2009). These regions get independently transferred to the nuclear genome of infected plant cells. The TL-DNA contains about 18 potential genes, of which 4 genes, rol A, B, C, and D, are implicated to induce the formation of hairy root in plants (Nemoto et al. 2009). The rolA gene suggested as an activator of growth and secondary metabolism while rolB gene as stimulator or growth-suppressor (Bulgakov 2008). The rolC has self-activation property and reported to play a significant role in hairy root growth (17-fold increase); however, rol A, B, and C together had 75-fold increase in Atropa belladonna (Bonhomme et al. 2000), while the TR-DNA possesses the genes for opine synthesis and the genes involved in the auxin biosynthesis, i.e., aux1 and aux2. The synergistic function of rolB, rolC, ORF13, and ORF14 of TL-DNA of A. rhizogenes in hairy root induction in Nicotiana tabacum and the effect of these genes on the rolB-mediated rooting were in the order ORF13 > rolC≤ORF14 (Aoki and Syono 1999). The Ri plasmid of R. rhizogenes strain has rolC and aux1 genes present on T-DNA designated as TL-DNA and TR-DNA, respectively. The detection of these genes in the hairy roots of host is indicative of T-DNA integration into the host genome. The virulence (vir) loci lying outside the T-DNA encode trans-acting products involved in early events in the plant-pathogen interaction. The vir loci are composed of six tightly regulated transcriptional units, virA, virB, virC, virD, virE, and virG, in different Agrobacteria. The virD2 gene is localized outside the T-DNA of Ri-plasmid, and this feature serves as diagnostics for the presence of any leftover Agrobacteria in the root tissue (Thiruvengadam et al. 2016). These genes play a role in inducing the expression of defense genes, thereby eliciting the production of secondary metabolite in and development of transformed roots.

9.3 Establishment of Hairy Root Cultures

9.3.1 Induction of Hairy Roots

The hairy root induction is affected by the type of strains of A. rhizogenes, co-cultivation period, type of explants, media composition, and PGRs. We have investigated these factors in our earlier study (Singh et al. 2020) and discussed here along with updated information available in literature, in brief.

9.3.1.1 A. rhizogenes Strains

The virulence of the A. rhizogenes strains has been observed to vary strain-wise, and it has effect on the frequency of the hairy root formation. In our experiment (Singh et al. 2020), in S. bryopteris the strain LBA 1334 was found to induce hairy root formation, while other two MTCC series strains (MTCC 532 and MTCC 2364) could not. Perhaps, it was believed that the recalcitrance in S. bryopteris to genetic transformation and the virulence of these strain affected the hairy root formation. Kim et al. (2015) reported HRC of Silene vulgaris by infecting leaf explants with A. rhizogenes strains, LBA9402, R1000, A4, 13333, and 15834, and the strain LBA9402 had induced the most hairy roots per plant. In Withania somnifera, the hairy root induction by above strains of A. rhizogenes (MTCC 2364, MTCC 532, R1000) showed the strain R1000 to be highly virulent in inducing hairy roots (50.6%), in comparison to MTCC 2364 (29.3%) and MTCC 532 (18.6%) (Chandrasekaran et al. 2015). Explants infected by MTCC 2364 and MTCC 532 showed maximum 77.6% and 67.6% of hairy root induction, respectively (Balasubramanian et al. 2018). Among different strains (R1000, 15834, and A4), R1000 was the most promising for hairy root stimulation as it was found to induce the highest growth rate, root number, root length, and transformation efficiency in Fagopyrum tataricum (Thwe et al. 2016). Joseph Sahayarayan et al. (2020) investigated effects of A. rhizogenes strains such as 15834, 13333, A4, R1200, R1000, LBA9402, R1301, and R1601 hairy root induction in Cucumis anguria. Their finding also supported the R1000 strain of A. rhizogenes as the most virulent and the best strain for hairy root initiation of C. anguria from cotyledon explants. Yousefian et al. (2020) reported hairy root induction in Mentha spicata by direct injecting of explants with A. rhizogenes strains (A13, R318, ATCC15834 A4, and GMI 9534). They found that of these four strains, the strain A13 exhibited the highest transformation efficiency (~75%).

9.3.1.2 Co-Cultivation Period

The co-cultivation period during transformation of explants with the R. rhizogenes strain has also shown to impact the hairy root formation. In S. bryopteris, the effect of co-cultivation period was examined for hairy root induction with the strain LBA1334 grown along with explants for 24 and 48 h (Singh et al. 2020). It was observed that the hairy root appeared after 6 days of infection with this strain in case of 48 h co-cultivation only. In Raphanus sativus, co-cultivation of explants with A. rhizogenes strain in half-MS medium containing acetosyringone (100 μM) for 2 days had the maximum effect on hairy root induction (Balasubramanian et al. 2018).

9.3.1.3 Type of Explants

Hairy roots usually induced in explants such as leaf, cotyledon, hypocotyl, node, stem, and root within 1–4 weeks of culture. Singh et al. (2020) reported hairy root induction after 6 days of infection using A. rhizogenes strain LBA1334 co-cultivated with root network as explants for 48 h after transformation. Yousefian et al. (2020) found that the middle and lower internodes of stem were highly susceptible to infection by A. rhizogenes, and these showed a higher rate of transformation. In Cucumis melo, cotyledon as explants was observed to give higher frequency of hairy root formation (Pak et al. 2009).

9.3.1.4 Media

The media compositions do affect the hairy root formation. Singh et al. (2020) tested different media like MS, SHFR (Stag Horn Fern Rooting), and Knops during transformation for hairy root induction by A. rhizogenes and found that SHFR plus TDZ (2 mg/L) and Bavistin (0.1%) showed good response in transformation and hairy root formation. The culture media incorporated with acetosyringone (an amino acid-derived phenolic compound) utilized by the invading Agrobacterium for food enhances the transformation rate by inducing the expression of vir gene (Veluthambi et al. 1989). Rana et al. (2016) reported the effect of medium supplements (30 g L−1 sucrose, 0.1 g L−1 l-glutamine and 5 g L−1 polyvinylpolypyrrolidone) on A. rhizogenes-mediated hairy root induction in Camellia sinensis var. sinensis. In HRC of A. indica, different media varying in ionic strength such as Ohyama and Nitsch (ON), Gamborg’s B5, and MS basal were used that yielded maximum amounts of biomass and azadirachtin in correlation with their ionic strength (Satdive et al. 2007). As ON medium contained the higher ionic concentrations of inorganic salts than the MS and B5, it was found to favor the growth of hairy roots as well as azadirachtin production.

9.3.1.5 PGRs

Though hairy roots have inherent ability to grow even in the absence of PGRs, the addition of PGR elicits the secondary metabolite production. Singh et al. (2020) reported SHFR media modified with PGRs in combination or alone (Kinetin, TDZ and Bavistin). The SHFR media with TDZ (2 mg/L) and Bavistin (0.1%) was found to increase the transformation efficiency and propagation of hairy roots (65%), while with Kinetin (2 mg/L) alone the response was 40% only. The phytohormone treatment, 0.3 mg L−1 IBA and 100 μM MeJA, in M. spicata was reported to substantially increase in the hairy root growth and phenolic acid accumulation (Yousefian et al. 2020). Joseph Sahayarayan et al. (2020) found that MS medium supplemented with IBA + NAA (2.46 + 1.07) had the maximum accumulation of biomass (0.68 g/L dry wt. and 6.52 ± 0.49 g/L fresh wt.) in 21-day-old transgenic hairy roots of Cucumis anguria. Methyl jasmonate elicited the biosynthesis of triterpenoid saponins in the hairy roots of S. vulgaris leading to increased level of segetalic acid (fivefold) and gypsogenic acid (twofold) than control hairy root (Kim et al. 2015). In transgenic hairy root line of Taxus x media var. Hicksii carrying a taxadiene synthase gene, the paclitaxel production and phenyl ammonia lyase activity upon elicitation with nitric oxide and methyl jasmonate yielded the highest paclitaxel content (7.56 mg L−1) (Sykłowska-Baranek et al. 2015).

9.3.2 Molecular Confirmation of Hairy Roots

The integration of Ri T-DNA into the genome of host plant cells causes the induction of hairy roots. The Ri T-DNA contains rol and aux genes, so these genes also integrate into the host genome along with T-DNA. The transgenic nature of hairy roots could be confirmed by simply amplifying these genes by PCR using rolC and aux1 gene primers. While the non-transgenic hairy root line can be detected by amplifying the virD2 gene present in in Ri-plasmid but not on T-DNA, it could be used as a diagnostic marker for the presence of any remaining Agrobacteria in the root tissue (Thiruvengadam et al. 2016). In S. bryopteris, rolA and virC genes were PCR-amplified from the hairy roots indicating that the hairy roots developed were due to T-DNA integration and not due to any A. rhizogenes present (Singh et al. 2020).

9.4 Productions of Secondary Metabolites in Using Hairy Root

HRC-based production of several plant secondary metabolites with known bioactivities such as shikonins, stilbenes, lignans azadirachtin, camptothecins, paclitaxel, hyoscyamine, harpagide, ginsenosides, and scopolamine was reported (Sim and Chang 1993; Satdive et al. 2007; Almagro et al. 2015; Zhang et al. 2017; Balasubramanian et al. 2018; Singh et al. 2020; Barba-Espin et al. 2020; Wawrosch and Zotchev 2021; Table 9.1; Fig. 9.1).

Fig. 9.1
14 chemical structures arranged in 6 rows as follows. Row 1, Shikonins, strictosidine, and catharanthine. Row 2, tabersonine, curcumin, and tanshinones. Row 3, anthocyanin and azadirachtin. Row 4, camptothecins and harpagide. Row 5, ginsenosides and paclitaxel. Row 6, hyoscyamine and scopolamine.figure 1

Chemical structures of some of the well-known secondary metabolites produced in HRC

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9.4.1 Shikonin

The HRC of Lithospermum erythrorhizon was reported long back in 1991 by Shimomura et al. (1991) using in vitro grown shoots for transformation with A. rhizogenes strain 15,834. They found that the hairy roots cultured on MS solid medium failed to produce any red pigments; however, in solid or liquid root culture media, a large amount of red pigments was produced. Further, they added adsorbents to the culture medium that stimulated shikonin production by approximately threefold. In Echium plantagineum, the hairy root lines developed using A. rhizogenes strain ATCC15834 (Fu et al. 2020) showed significant difference in the biomass and shikonin production in the 1/2B5 and M9 media. It was observed that the biomass in the 1/2B5 medium was fivefold than the M9 and the content of acetylshikonin was twofold in the 1/2B5 medium (36.25 mg/L on average) than the M9 medium.

9.4.2 Phenolic Compounds of HRC

Several phenolic compounds such as rosmarinic acid, caffeic acid, chlorogenic acid, lithospermic acid B, and cinnamic acid were observed in the hairy roots of M. spicata, developed by infection with different A. rhizogenes strains (ATCC15834, A13, A4, 9534, and R318), and their content was compared (Yousefian et al. 2020). Ho et al. (2018) reported HRCs of Polygonum multiflorum from leaf explants infected by A. rhizogenes strain KCCM 11879 with more than 60% transformation efficiency after 21 days of co-cultivation. It was found that the line HR-01 yielded the highest biomass (9.7 g L−1 of DW) and total phenolic content (26.64 mg g−1 DW).

9.4.3 Triterpenoid Saponins

Phytochemical studies of Gynostemma pentaphyllum revealed nearly 90 dammarane-type saponin glycosides, known as gypenosides, that show pharmacological activities (Yin et al. 2004). HRC of G. pentaphyllum was established by infecting leaf discs with A. rhizogenes (Chang et al. 2005); it yielded 7.3 g L(−1) the dry wt. of biomass in MS medium for a period of 49 days with a gypenoside content of 38 mg g(−1) dry wt. Kim et al. (2015) also reported the in vitro production of triterpenoid sapogenins in HRC of S. vulgaris by infecting leaf explants with five strains of A. rhizogenes.

9.4.4 Artemisinin

Mass cultivation of hairy roots in Artemisia annua was reported in a modified 3-L stirred tank bioreactor using optimized culture conditions (Patra and Srivastava 2014). In this bioreactor, it was possible to produce biomass, 18 g L(−1) (dry wt.), and artemisinin content, 4.63 mg L(−1), in a period of 28 days, which further increased to 10.33 mg L(−1) in response to methyl jasmonate. Elicitation of artemisinin production with 150 mg chitosan l(−1) in hairy roots of Artemisia annua was reported to be increased sixfold to 1.8 microg mg(−1) dry weight in 6 days (Putalun et al. 2007). Wang et al. (2006) reported use of oligosaccharide elicitor (MW < 2500) from an endophytic fungus, Colletotrichum gloeosporioides, for the stimulation of artemisinin production in hairy roots of A. annua. They observed that the 23-day-old hairy roots on exposure to the elicitor at 0.4 mg/mL for 4 days yielded the maximum artemisinin content, 13.51 mg/L (51.63% increase over the control). Further, it was also reported that the nitric oxide generated by an oligosaccharide elicitor from Fusarium oxysporum mycelium potentiates its role in induction of artemisinin production in A. annua hairy roots (Zheng et al. 2008). The combination of sodium nitroprusside (NO donor) with OE increased artemisinin content from 1.2 to 2.2 mg/g dry wt., whereas the content of artemisinin in HRC was 28.5 mg/L, a twofold increase over the OE treatment alone. Tetraploid clones of Artemisia annua hairy roots produce more artemisinin (six times) than diploid parent (De Jesus-Gonzalez and Weathers 2003). The physical and chemical factors such as light, pH value 5.4 of the medium, and 3% sucrose in the medium along with gibberellin (4.8 mg/L) had effect on the growth of the hairy roots and production of artemisinin (Cai et al. 1995).

9.4.5 Paclitaxel

Paclitaxel (trade name: Taxol®) is a potent anticancer agent extracted from the yew plants (Taxus spp.). Due to its low accumulation levels in the plants of Taxus spp., the yield of paclitaxel is limited and could not meet the demand for the pharmaceutical use. Therefore, alternative methods including HRC have been attempted for in vitro production of paclitaxel from Taxus spp. (Kim et al. 2009; Sykłowska-Baranek et al. 2015; He et al. 2022). HRCs have been reported in different Taxus spp. such as of T. cuspidate (Korean yew) (Kim et al. 2009), Taxus x media var. Hicksii (Sykłowska-Baranek et al. 2015); and T. baccata (He et al. 2022) using A. rhizogenes. Kim et al. (2009) reported that methyl jasmonate treatment of the hairy root line (RC11106) led to accumulation of 52.5 mg/L of taxol over 2 weeks of incubation. Paclitaxel production in HRC of Taxus x media var. Hicksii with a taxadiene synthase transgene upon elicitation by nitric oxide and methyl jasmonate enhanced the paclitaxel content to 7.56 mg L−1 after 2 weeks of treatment (Sykłowska-Baranek et al. 2015).

9.4.6 Azadirachtin

Azadirachtin is a limonoid (C35H44O16, tetranortriterpenoid) and the major component of widely used neem (Azadirachta indica)-based biopesticides. In HRC of A. indica, the effect of ON, Gamborg’s B5, and MS basal media was investigated on yield of biomass and azadirachtin content (Satdive et al. 2007). Out of the three media, ON medium, that contained higher ionic concentrations of inorganic salts than the MS and B5 media, favored the growth and azadirachtin production (0.0166% dry weight, DW). Further, addition of biotic elicitor increased the production of azadirachtin by nearly fivefold (0.074% DW), while jasmonic acid and salicylic acid showed an approximately six- (0.095% DW) and ninefold (0.14% DW) increase, respectively, on ON medium (Satdive et al. 2007). The mass culture of hairy roots of A. indica in gas-phase reactors (nutrient spray and nutrient mist bioreactor) yielded the biomass 9.8 g/L dry wt. and azadirachtin accumulation of 2.8 mg/g (Srivastava and Srivastava 2012).

9.4.7 Camptothecin

Camptothecin, an important anticancer drug, is believed to be a potent topoisomerase inhibitor that interferes with the topoisomerase in DNA replication HRC of Ophiorrhiza alata which was developed by infection of nodal explants of in vitro-grown plant with A. rhizogenes TISTR 1450 for camptothecin (CPT) production (Ya-ut et al. 2011). The content of CPT in various parts of O. alata was analyzed by HPLC and found to be double in transformed hairy roots (785 ± 52 μg/g dry wt) than in the soil-grown plants (388 ± 32 μg/g dry wt). Kamble et al. (2011) reported hairy root induction in O. rugosa, another source of CPT, using A. rhizogenes strain LBA9402. The CPT contents in the hairy roots and in vitro-grown transformed shoots were 0.009% d.w. and 0.012% d.w., respectively.

9.4.8 Ginsenoside

Ginseng (Panax ginseng) roots contain ginsenosides (saponins), utilized for its bioactive properties such as immunomodulatory, hepato- and cardioprotective, stamina booster, antifatigue, and physiological and pharmacological effects. Yu et al. (2005) investigated the impact of temperature and light quality on biomass and ginsenoside content of HRC in the bioreactor. They observed that biomass of hairy roots was highest under dark or red light, while ginsenoside content was optimum under fluorescent light. Kochan et al. (2013) reported three independently generated hairy root (A, G, and B) of P. quinquefolium upon infection by A. rhizogenes. It was found that the line A had the highest increase of dry biomass (above eightfold) followed by line G (sevenfold) and line B (fivefold) for the period of 28 days culture. In the developed hairy roots of P. quinquefolium, the total ginsenoside level in hairy root lines A, G, and B were recorded to be about 10, 8, and 6 mg/g dw, respectively (Kochan et al. 2013).

9.4.9 Hyoscyamine

In Datura stramonium, HRC developed with hairy roots of different ploidy (diploid and tetraploid) levels was investigated by Pavlov et al. (2009) for hyoscyamine biosynthesis. The ploidy level difference was observed in content of hyoscyamine and the influence of nutrients growth of hairy root. The HRC of tetraploid plants had the maximum yield of hyoscyamine (177 mg/L) in MS medium with 6% sucrose.

9.4.10 Scopolamine

Scopolamine is a tropane alkaloid that shows anticholinergic property. The overexpression of genes of scopolamine biosynthesis putrescine N-methyltransferase (PMT) and hyoscyamine 6 β-hydroxylase (H6H) in Hyoscyamus niger’s transgenic HRC produced significantly higher (P < 0.05) levels of scopolamine in comparison to the wild-type and transgenic lines harboring pmt or h6h gene alone (Zhang et al. 2004). The line (T3) overexpressing the genes, pmt and h6h, was observed to produce the highest content of scopolamine (411 mg/L) followed by single-gene (h6h) transgenic line H11 (184 mg/L) and wild type (43 mg/L). Dehghan et al. (2017) found that the tetraploidy improved the overexpression of h6h and scopolamine production of H. muticus.

9.4.11 Harpagide

Harpagide is an iridoid glycosides that show anti-inflammatory properties. Only a few literature are available on the hairy root line development for harpagide production. Piątczak et al. (2019) reported HRC in Rehmannia elata (Orobanchaceae) from shoot tips and leaves by the infection of A. rhizogenes strain (A4). They observed the different levels of iridoid glycoside content, in the hairy root line (S1 line) were aucubin (0.2 mg/g DW) and harpagide (1.57 mg/g DW), and in L14 line, harpagoside (0.09 mg/g DW) were the highest.

9.5 Upscaling the Productions of Secondary Metabolites in Bioreactors Using HRCs

For scaling up the production of secondary metabolites and biomass of hairy roots, a large vessel (bioreactor) is needed that can provide the best conditions for optimum growth and secondary metabolite production. But the delicate and interconnected form of the hairy roots makes the growth measurement and designing of a large-scale culture system a difficult task. Stiles and Liu (2013) have discussed the major factors related to large-scale bioreactor cultures such as process intensification technologies and the mathematical models and computer-aided methods of bioreactor design and development. Wyslouzil et al. (2000) suggested that the bioreactor design for HRC is a balancing act between the biological needs of the tissues without inducing an additional and undesirable biological response. The bioreactors used in HRC are usually (1) liquid-phase, (2) gas-phase, and (3) hybrid reactors.

Bioreactor designs for HRC are challenging and should meet the criteria such as least mechanical agitation (may cause wounding and shearing of delicate hairy roots leading to callus formation), uninterrupted nutrient flow (hampers due to branching and interconnected forms hairy roots), regular oxygen supply, nutrient uptake, etc. in the medium. An air-lift fermenter system along with a XAD-2 column was used for the shikonin production from HRC of L. erythrorhizon (Shimomura et al. 1991), which continuously produced ~5 mg/day of shikonin during a period of over 220 days. Srivastava and Srivastava (2012) reported azadirachtin production by hairy roots of A. indica using HRC in gas-phase reactors (nutrient spray and nutrient mist bioreactor). They found the biomass production of 9.8 g/L dry weight with azadirachtin accumulation of 2.8 mg/g biomass (27.4 mg/L) during 25 days of batch cultivation period.

The study by Zhang et al. (2004) provided an effective approach for large-scale commercial production of scopolamine by using transgenic hairy root culture systems as bioreactors. Homova et al. (2010) investigated the capacity of HRC of Harpagophytum procumbens to accumulate four phenylethanoids, viz., glycosides beta-OH-verbascoside, verbascoside, leucosceptoside A, and martynoside in shake flasks and a stirred tank reactor. They observed equally highest contents of verbascoside in both kinds of culture (1.12 mg/g dry weight), while leucosceptoside A content was 1.6 times higher in bioreactor than in shake flask. Several reviews are available discussing bioreactor-based scale-up using HRCs (Valdiani et al. 2019).

9.6 Elicitors Increase the Secondary Metabolite Production in HRCs

Various biotic and abiotic elicitors are used in HRCs to elicit the biosynthesis and the accumulation of secondary metabolites (Yousefian et al. 2020; Singh et al. 2020; Joseph Sahayarayan et al. 2020). These elicitors are known to activate different genes such as secondary metabolic pathway, defense-related or signaling pathway, and transcription factors. In general, biotic elicitors used in HRC include fungal cell extracts, fungal or plant polysaccharides, and heavy metals, while the abiotic elicitors include PGRs (auxins, TDZ, cytokinins), signaling molecules (nitric oxide, hydrogen peroxide, salicylic acid, methyl jasmonate), UV radiation, ultrasound, shaking, and temperature. For the best result on elicitation of in vitro production of metabolites in the medium, the selection of suitable elicitor, its level to be added, and treatment duration need to be standardized. The elicitor treatments like other tissue culture methods also stimulate the production and release of intracellular products in the hairy roots.

9.7 Prospect and Challenges

HRC-based in vitro culture is a promising approach for production of a wide range of secondary metabolites as compared to other in vitro methods such as cell culture and adventitious root culture (Table 9.2). Several factors critical to increase in yield of HRC have been standardized such as light, oxygen, temperature, phytohormones, heavy metals, elicitors, external stimuli, etc. The metabolic pathway engineering for modulation of expression of key genes either by overexpression of genes and transcription factors or by downregulation of competing pathway genes has also led to increased yield of end-products. With advances in genomics and molecular biology, today we have a better understanding of the biosynthetic pathway and their regulation, thus realizing that the biosynthetic potential of hairy roots is more feasible. Commercial production of secondary metabolites that have phytoceutical uses, from field-grown plants, can’t meet the market demand, for example, the extraction of catharanthine and vindoline from field plants and then chemical semi-synthesis of vinblastine and vincristine, two chemicals usually found in the FDA drug shortage list due to supply chain problems (Kaiser 2011). Due to complex chemical structures, most of secondary metabolite economic feasibility for the industrial mass production through chemical synthesis is not possible and thus not popular as method of choice.

Table 9.2 Comparison of hairy root culture with other tissue culture methods
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Despite vast potential and prospect of HRCs, very few companies (public or private sector) have initiated the HRC-based commercial production phytochemicals from hairy roots or any in vitro methods unlike a large number of companies worldwide involved in the mass production of tissue culture raised plants. ROOTec bioactives Ltd. (www.rootec.com) is the only company founded in 2005 in Switzerland for the commercial production of plant-derived molecules using bioreactor technology to provide products for the pharmaceutical and cosmetic markets. Besides in vitro production of secondary metabolites, the hairy roots have been useful for the functional validation of genes, plant synthetic biology, genome editing, symbiotic root colonization, antimicrobial excretion, and phytoremediation (Morey and Peebles 2022). Deciphering the competing biosynthetic pathways or identifying feedback or feed-forward reactions in metabolite production and then devising strategies is important for increasing the yield desired product. The major hurdle in commercial exploitation of HRC is the development of suitable protocol for the sustainable growth of hairy roots and designing of appropriate bioreactors for scaling-up without damaging the delicate hairy roots. Metabolic engineering for manipulation of biosynthetic pathways for high yield of secondary metabolites is easier nowadays with the availability of vast genomic and transcriptomic datasets helping better understanding of regulation of pathways.

9.8 Conclusion

Among various tissue culture methods, HRC has the potential to be harnessed for in vitro production of secondary metabolites at commercial level. It could be an economically viable option for sustainable in vitro production of secondary metabolites, as the hairy roots are normally high yielding due to their fast growth rates, possible growth in hormone-free media, and genetic and biosynthetic stability. These are also amenable to genetic manipulation using genetic engineering including recent gene-editing approaches. In future, the HRC efficacy and commercial viability could be enhanced by adopting advanced biochemical processing methods for metabolite extractions, genetic manipulations, and elicitations for enhanced yield and bioreactor-based up-scaling.