Compounds from medicinal plants produced in hairy root and transgenic hairy root cultures: a review

Phenolic compounds produced in hairy roots

The phenylpropanoid pathway synthesizes phenolics, which have a phenol group as their base structure. Their precursors are the aromatic amino acids phenylalanine and tryptophan (Tian, 2015). Phenols are classified as phenolic acids, xanthones, flavonoids, lignans, coumarins, naphthoquinones, and tannins (Lattanzio, 2013). Plants produce these compounds as a defensive response to pathogen organisms (Tashackori et al., 2016). Numerous reports demonstrate the antioxidant, anticancerogenic, anti-inflammatory, antiviral, and antimicrobial activity of phenolic-rich extracts or pure compounds, making them of pharmacologic interest (Weremczuk-Jezyna et al., 2013; Bazaldúa et al., 2019; Fu et al., 2020; Zheleznichenko et al., 2018; Chung et al., 2016). HR cultures have the capacity to produce a variety of phenolic compounds. Table S1 lists the medicinal phenolic compounds discovered over the past 14 years.

Bioactive terpenes produced in hairy roots

Mevalonic acid and methylerythritol phosphate pathways synthesize terpenes from isoprene unities, with dimethylallyl diphosphate and isopentenyl diphosphate serving as their precursors (Tian, 2015). Terpenes are classified as the number of isoprene units in the structure; the most common are monoterpene, diterpene, triterpene, and tetraterpene (Xavier et al., 2023). These compounds are important to the plant because they play a role in primary processes of metabolism, such as photosynthesis, respiration, growth, and cell membrane formation. Moreover, some of these compounds integrate essential oils, others are defensive compounds, and a few are plant pigments of leaves and fruits (Pulido, Perello & Rodriguez-Concepcion, 2012). Pharmacological activities reported for terpenes are antiviral, antimicrobial, anticancerogenic, anti-inflammatory, anxiolytic, and hepatoprotective, among others (Kohsari et al., 2020; Khazaei et al., 2019; Moreno-Anzúrez et al., 2017; Huang et al., 2014; Torkamani et al., 2014; Mahesh & Jeyachandran, 2011). Table S2 presents the progress made over the past 14 years in producing terpenes of medicinal importance through hairy roots.

Medicinally important alkaloids produced in hairy roots

Alkaloids exhibit intriguing properties because their water solubility is high when the media is acidic, but they are lipid soluble in neutral and basic conditions (Heinrich, Mah & Amirkia, 2021). This property is crucial for understanding pharmacokinetics across membranes. The continuous search for new natural products with pharmacological effects is still mandatory. Even more, the use of natural products could offer an advantage over synthetic compounds, since they could be more easily recognized as substrates by active transporters (Heinrich, Mah & Amirkia, 2021). Alkaloids are compounds that are synthesized in secondary metabolism pathways and have a chemical structure that includes at least one nitrogen atom.

According to the precursor molecules that provide the nitrogen atom to form alkaloid structure, they can be divided into three groups: true alkaloids, protoalkaloids, and pseudoalkaloids (Gutiérrez-Grijalva et al., 2020). True alkaloids have the nitrogen atom in a cyclic structure, and the precursor molecules are the amino acids L-ornithine, L-tyrosine, L-phenylalanine, L-lysine, L-histidine, L-tryptophan, L-arginine, and glycine/aspartic acid. In protoalkaloids, the nitrogen atom is outside of the ring; the precursors are the amino acids L-tyrosine and L-tryptophan or biogenic amines (they are rare in nature). The last group is pseudoalkaloids, where the nitrogen atom is part of a heterocycle in the molecule, but the precursors are acetate, pyruvic acid, adenine/guanine, or geraniol (Gutiérrez-Grijalva et al., 2020).

Alkaloids, as other secondary metabolites used as medicines, have been related to plant defense since they protect plants from predators (Chik et al., 2013). Around 12,000 alkaloids have been reported, and they are synthesized from different metabolic pathways like shikimate, ornithine lysine, nicotinic acid, histidine, purine, terpene, and polyketides (Schläger & Dräger, 2016; Gutiérrez-Grijalva et al., 2020). According to their chemical structure, they are classified as pyrrolidines, indolizines, quinolines, tropanes, terpene alkaloids, and aromatic alkaloids, among others (Brielmann et al., 2006). The biological activity reported for these compounds is antitumoral, analgesic, anti-inflammatory, antimicrobial, and antihypertensive (Ya-ut, Chareonsap & Sukrong, 2011; Dehghan et al., 2012; Sharifi et al., 2014; Hanafy et al., 2016; Thakore, Srivastava & Sinha, 2017).

The most well-known alkaloids used against cancer are camptothecin, vinblastine, vincristine, and vindesine. Other well-known alkaloids used in medicine include morphine, quinine, ephedrine, and nicotine (Kurek, 2019). The use of HR induced by transformed R. rhizogenes strains with specific constructions is a great opportunity for the development of this field research (Heinrich, Mah & Amirkia, 2021). Table S3 displays the alkaloids of medicinal importance that have been produced in hairy root cultures over the past 14 years.

Hairy roots synthesize a major concentration of plant bioactive compounds

Hairy root cultures typically produce more bioactive molecules than those found in the organs of wild plants (Pala et al., 2016; Tuan et al., 2018; Yoon, Chung & Thiruvengadam, 2015). Mishra et al. (2011) reported a higher production of picrotin and picrotoxinin in HR cultures of Picrorhiza kurroa Royle ex Benth. They observed that the hairy roots induced with the A4 strain produced 13 times more picrotin (8.81 g/g DW) and 6 times more picrotoxinin (47.1 g/g DW) compared to the wild plant. Nagella et al. (2013) established the HR culture of Gymnema sylvestre R. Br. to produce gymnemic acid; they observed that the HR production was 11.3 mg/g DW, which corresponds to 4.7 times more than the production of wild roots.

In another study, Weremczuk-Jezyna et al. (2013) reported the production of rosmarinic acid (RA) by HR lines culture of Dracocephalum moldavica L. They analyzed the increase of this compound at different culture medium conditions. They evaluated three culture media, its concentration, and the light/darkness effects (Weremczuk-Jezyna et al., 2013). The highest accumulation of RA (78.08 mg/g DW) was obtained from HR grown in B5 culture medium at 50% of nutrients and in the photoperiod (16/8 light/dark). The accumulated RA represents 9.9 times more than its production in wild plants.

Moreover, Samadi et al. (2014) established the HR culture of Linum mucronatum ssp. mucronatum to produce podophyllotoxin (PTOX) and 6-methoxy podophyllotoxin (6-MPTOX). They observed the production increment of both compounds five times (PTOX 5.78 mg/g DW and 6-MPTOX 49.19 mg/g DW), compared with their production in wild root cultures (Samadi et al., 2014). On the other hand, Balasubramanian et al. (2018) evaluated two R. rhizogenes strains, MTCC 2364 and MTCC 532, to induce HR in Raphanus sativus L., with the MTCC 2364 strain showing the major response to hairy root development. Regarding the quantification of phenolic compounds and antioxidant activity in HR, they reported an increase of 33.07 mg/g of gallic acid equivalents (GAE) for phenols and 48.03 mg/g of quercetin equivalents (QE) of flavonoids, which were 1.19 and 1.22 times higher, respectively, compared with wild roots. This influenced biological activity since the hairy root extract showed a major antioxidant response.

In addition to the HR culture, Grzegorczyk-Karolak et al. (2018) reported the Salvia viridis L. HR culture in Woody Plant Medium (WPM). They evaluated biomass and phenolic compounds increasing when the HR was developed in photoperiod (16 h light/8 h dark) and darkness. When biomass and phenols increased in darkness conditions, the major compound was rosmarinic acid with 35.8 mg/g DW in the K3 line, which corresponds to 8 times higher when compared to that reported on wild plant roots (Grzegorczyk-Karolak et al., 2018). By comparison, Matvieieva et al. (2019) started the culture of Artemisia vulgaris L. to produce phenolic compounds. They obtained twelve lines of hairy roots, four of which accumulated a higher concentration of flavonoids (65–73 mg of rutin equivalent, which corresponds to 1.3 and 1.5 times more than the control groups), and nine of these lines exhibited significant antioxidant activity compared to the organs of the wild plant.

Vinterhalter et al. (2019) induced THR in Gentiana utriculosa L. to produce bioactive xanthones. They analyzed the chemical profile of hairy root line extracts and reported that one of them accumulated a higher concentration of decussatin (4.54 mg g DW) and decussatin-1-O-primeveroside (8.13 mg g DW), which represents 2.2 and 1.1 times more when compared to wild root production (Vinterhalter et al., 2019). In another study, Zheleznichenko et al. (2018) reported the culture of HR of Nitraria schoberi L. and observed that the hairy roots produced a higher quantity of flavonoids, hydroxycinnamic acids, and saponins. They also reported that the HR extracts had higher antiviral activity than Tamiflu. Sahai & Sinha (2020) induced HR on in vitro cultures of Taxus baccata subsp. wallichiana to produce taxol. They observed that the hairy roots produced 12 mg/g DW, which is 80 times more than wild plants.

Yousefian, Golkar & Mirjalili (2021) evaluated two R. rhizogenes strains (LBA-9402 and C58C1) and one modified A. tumefaciens C58C1 (pRiA4) strain to induce HR in Pelargonium sidoides DC. for obtaining the antiviral coumarin, umckalin. Interestingly, only the modified A. tumefaciens induced THR. They obtained the highest production of umckalin (228.98 µg/g DW) in THR cultures elicited with 100 µM MeJa, which corresponds to 1.61 times more than in the THR cultures without the elicitor. On the other hand, Alamholo & Soltani (2023) established the HR culture of Thymus daenensis Celak to produce phenolic compounds. The HR were induced with the ATCC 15834 R. rhizogenes strain and produced more phenols (209.28 mg gallic acid/g) and flavonoids (4.29 mg quercetin/g) than wild roots, which corresponds to 2.79 and 1.55 times, respectively. Elsewhere, Zhao et al. (2023) induced HR in Plumbago auriculata L. using three R. rhizogenes strains (A4, ATCC 15834, and LBA 9402) for obtaining plumbagin. Three HR lines high producers were selected; the highest yield was obtained with the line PAHR 15834 (38.95 mg/g DW), followed by PAHR 9402 (24.90 mg/g DW) and PAHR 4 (24.72 mg/g DW), which corresponds to 72.13, 46.11, and 45.22 times more than obtained in wild roots.

Hairy roots produce compounds that are accumulated in the leaves of wild plants

Unlike suspension cells or callus cultures, which are dedifferentiated cells (Ochoa-Villarreal et al., 2016), the hairy root culture contains differentiated cells that can synthesize specific compounds produced in complex metabolic pathways such as terpenoid indole alkaloids (Morey & Peebles, 2022). The organelle-compartmentalized plant cell synthesizes certain terpene types, such as monoterpenes, diterpenes, and tetraterpenes. They are synthesized through the methylerythritol phosphate pathway, which is active in plastids mainly found in photosynthetic tissues (Gangwar, Kumari & Jaiswal, 2022). In this regard, Wang, Zhou & Zhang (2012) established an HR culture of Siegesbeckea orientalis L. to produce kirenol, a diterpene that accumulates in the leaves of wild plants. They observed that the hairy roots produced 1.6 mg/g DW of the compound, which is five times more than the roots of a wild plant but twice as little as reported in leaves. Even though HR showed a lower concentration of kirenol than wild plant leaves, hairy roots were able to produce the compound in a stable and continuous manner.

Hanafy et al. (2016) established THR cultures of Catharanthus roseus to produce vincristine and vinblastine. The highest yield of vincristine (442.3 ng/mg FW) was obtained in THR grown in liquid MS medium and was 1,474 times higher than wild roots, but it was not produced when THR was grown in solid MS medium (Hanafy et al., 2016). On the other hand, THR cultures grown in liquid MS medium produced a low amount of vinblastine (two ng/mg FW), while THR grown in solid MS medium produced 6 times more (12 ng/mg FW), though it was 1.33 times lower than obtained in wild roots. Another interesting result was the catharanthine production by THR (0.7 ng/mg FW in half liquid medium MS), which was not produced by wild roots. Furthermore, two months after the establishment of the culture, the secretion of the compounds into the liquid medium revealed a concentration of 1.5 ng/mL for vincristine, 8.5 ng/mL for vinblastine, and 16.5 ng/mL for catharanthine.

Hairy root cultures can be elicited to increase the production of plant bioactive compounds

In hairy root cultures, changes in culture conditions affect plant bioactive compound production (Bazaldúa et al., 2019). Changing the culture medium, temperature, and adding precursors and exogenous molecules can change the chemical profile and growth index. This can sometimes promote the synthesis and accumulation of compounds of medicinal interest (Grzegorczyk-Karolak et al., 2018; Renouard et al., 2018; Ruffoni et al., 2016; Tuan et al., 2018).

Hairy roots synthesized de novo compounds

Production of de novo compounds was reported in HR cultures. Tusevski et al. (2013) established the HR cultures of Hypericum perforatum L. and reported differences in the chemical profile of hairy root lines and identified four new xanthones: 1,3,5,6-tetrahydroxyxanthone, 1,3,6,7-tetrahydroxyxanthone, γ-mangostin, and garcinone C. On the other hand, Cong et al. (2015) reported for the first time two glycosylated aryltetralin lignans, the 4′-demethyl-methoxypodophyllotoxin glucoside and the demethyl-deoxypodophyllotoxin glucoside in hairy roots of Linum album and Linum flavum that had not been identified in Linum genus species.

Moreover, Moreno-Anzúrez et al. (2017) established a transgenic hairy root culture of Lopezia racemosa Cav., with the goal to produce anticancer and anti-inflammatory compounds. The bioactive fraction afforded the major compound (23R)-2α,3β,23,28-tetrahydroxy-14,15-dehydrocampesterol that was not reported previously.

Hairy root cultures scaled-up in bioreactors

Hairy roots are a system of culture that facilitates the scale-up to produce bioactive compounds using bioreactors. In this regard, Ionkova et al. (2013) established the HR culture of Linum narbonense L. to produce justicidine B and optimize the compound production using a BIOSTAT B Plus bioreactor. They found a yield of 7.78 mg/g DW in the culture flask and 7.89 mg/g DW in the bioreactor; the in vitro culture accumulation of justicidine B was 15 times higher than the accumulation in wild roots. Habibi et al. (2015) optimized scopolamine yield in Atropa belladonna L. HR grown in a bioreactor. The HR culture was established in a 1.5 L glass bioreactor with agitation. They reported the effect of agitation (40, 70, 110 rpm) and aeration (0.75, 1.25, 1.75 vvm) on the scopolamine production. Their highest scopolamine yield (1.59 mg/g DW) was at 70 rpm and 1.25 vvm.

Patra & Srivastava (2016) optimized the production of artemisinin in the HR culture of Artemisia annua. They compared the concentration of the interest compound in HR growth in three kinds of bioreactors. The highest concentration of artemisinin was achieved in a modified mist bioreactor (1.12 mg/g DW), while the yield obtained in a bubble column bioreactor was 0.27 mg/g DW. Nevertheless, the mist bioreactor without modifications yielded the lowest concentration, measuring 0.22 mg/g DW. On the other hand, Thakore, Srivastava & Sinha (2017) optimized the production of ajmalicine in HR of Catharanthus roseus by modifying the culture conditions. Additionally, they established cultures in various bioreactor systems and measured the compound production in biomass. The higher biomass (7.7 g/L) and ajmalicine (34 mg/L) production was achieved in HR growing in a bubble column bioreactor with a polyurethane support.

Vinterhalter et al. (2021) optimized the THR culture systems of Gentiana dinarica Beck for xanthone production. The THR lines cl-B, cl-D, cl-3 and cl-14 were cultured in half MS medium placed in Erlenmeyer flasks (EF), in the RITA^® temporary immersion system (TIS-RITA), and in a bubble column bioreactor (BCB). The xanthone norswertianin-1-O-primeveroside increased in THR (cl-D line) cultures grown in half MS medium added with 4% sucrose; however, there was not a significant difference from those cultures grown in EF or TIS-RITA. On the other hand, the highest norswertianin yield was in the THR cl-B line grown in the TIS-RITA system (18.08 mg/container), in culture with half MS medium added with 2% sucrose.

Kozlowska et al. (2024) established HR cultures of Agastache rugosa and optimized the bioreactor cultivation to increase the biomass, phenolic compounds, and RA production. The systems used were a nutrient spray bioreactor (NSB), two temporary immersion systems (Plantform^® and RITA^®), and 300 mL Erlenmeyer flasks (EF). The most efficient system for biomass production was RITA^® (15.96 g DW/L), followed by EF (10.24 g DW/L), NSB (8.71 g DW/L), and Plantform^® (2.91 g DW/L). While the highest yield of phenolic compounds was obtained with NSB, and the RA content in the NSB system was 9.16 mg/g DW, which is like that reported for the leaves of the wild plant (9.31 mg/g DW) (Zielińska et al., 2016).

Hairy roots secreted medicinal plant compounds into the culture media

One of the advantages of HR is the presence of specialized cells that have a complete endomembrane system that allows them to produce a great diversity of secondary metabolism molecules (Yoo et al., 2014). Wild plants synthesize some active compounds through metabolic pathways involving various cellular organelles such as vacuoles, cytosol, peroxisomes, chloroplasts and mitochondria. Synthesis and accumulation of metabolites can occur in cells and tissues of the same or different organs (Roze, Chanda & Linz, 2011). THR obtained from the expression of T-DNA genes from R. rhizogenes can produce complex secondary metabolites. In the case that the compounds have a different synthesis and accumulation site, HR cells can excrete the compounds into the culture medium or store them in specific organelles such as vacuoles (Hanafy et al., 2016).

When THRs excrete compounds, they can be easily recovered from the culture medium, allowing continuous production in the system. When the compounds are accumulated in vacuoles, there are specific products that permeabilize the membrane and cell wall of HRs to release them into the culture medium without affecting the viability of the HR (Kamiński, Bujak & Długosz, 2024). In this regard, Fu et al. (2020) established the HR culture of Echium plantagineum L. to produce shikonin derivatives. They reported the secretion of compounds into the culture medium and found that the WT3 line was a major producer of acetylshikonin (42.69 mg/L) in half B5 medium.

Reyes-Pérez et al. (2022) induced THR in Sphaeralcea angustifolia (Cav) G. Don to produce scopoletin and sphaeralcic acid. The SaTR N5.1 line had a growth index (GI) 5 times higher than the SaTR N7.2 THR line, but this one had the highest active compound production after 2 weeks of culturing. The scopoletin and sphaeralcic acid yields were 0.151 mg/g DW and 17.6 mg/g DW, respectively. Moreover, both compounds were secreted into the culture medium. The concentration of scopoletin (0.43 mg/L) in the culture medium was higher than the accumulated in the THR, while the concentration of sphaeralcic acid (15.5 mg/L) was similar to that in the THR.

Also, the HR lines of Calendula officinalis L. established by Długosz et al. (2013) were optimized by Kamiński, Bujak & Długosz (2024) to stimulate the excretion of oleanolic acid (OA) derivatives into the culture medium. For this, dimethyl sulfoxide (DMSO), Tween 20 (T₂₀), Tween 80 (T₈₀), and Triton X-100 (Tx100) were used as surfactants. The effect of each one of the surfactants in combination with ultrasound (US) to stimulate the permeabilization of the metabolite was analyzed. The T₂₀ (1%) was the most effective treatment, significantly increasing the content of OA derivatives in three HR lines resulting in production that were 11, 61 and 22 times higher than those of each control. The combination of US and T₈₀ or T₂₀ increased even more the OA derivatives yield.