Hairy Root Culture System Research Articles

Stilbene-rich extract increases the cytotoxic effects of paclitaxel in hormone receptor-positive and triple-negative breast cancer spheroids

BackgroundTriple-negative breast cancer (TNBC) is an aggressive form of breast cancer that lacks three receptors commonly found in breast cancer cells. It is associated with high mortality rates, and therefore, investigating therapies to increase survival rates is crucial. Plant-derived compounds are being explored as potential adjuvants for common chemotherapy drugs, such as paclitaxel (Pac). PurposeThe study aimed to evaluate the cytotoxic effect of a prenylated stilbene-rich extract (SRE) produced via a sustainable peanut hairy root culture system and observe its potential as an adjuvant for Pac in human triple-negative and hormone receptor-positive (HR+) breast cancer spheroids. The effects were compared to arachidin-1 (A-1), a cytotoxic prenylated stilbene present in the extract. MethodsSRE was produced from elicited peanut hairy root cultures. The extract was purified using chromatography techniques to obtain the prenylated stilbene arachidin-1 (A-1) with a purity of over 95 %. TNBC cell lines MDA-MB-231, MDA-MB-436, and HR+ breast cancer cell line MCF-7 were used to evaluate the cytotoxicity and apoptotic activity of SRE in comparison with A-1. Two-dimensional (2D) experiments were performed using cell viability assays and imaging microscopy. Three-dimensional (3D) spheroids cultures were established, and the impact of SRE alone and in combination with Pac on cell viability and caspase 3/7 activity was evaluated. ResultsSRE (10 μg/mL) inhibited cell proliferation by approximately 50 % in TNBC and MCF-7 cells in a time-dependent manner. Additionally, Annexin V FITC/PI staining revealed that SRE (10 μg/mL) induced more apoptosis than A-1 at the equimolar concentration (5 μM) in MDA-MB-231 cells. Combining SRE with Pac decreased spheroid cell viability and induced apoptosis by activating caspases 3 and 7 in TNBC and HR+ breast cancer spheroids. ConclusionsThese findings highlight the potential of SRE as a novel adjuvant for Pac chemotherapy in TNBC and HR+ breast cancer treatment.

Nanoparticle elicitation: A promising strategy to modulate the production of bioactive compounds in hairy roots

Hairy root culture is one of the promising biotechnological tools to obtain the stable and sustainable production of specialized metabolites from plants under controlled environment conditions. Various strategies have been adopted to enhance the accumulation of bioactive compounds in hairy roots yet their utilization at the commercial scale is restricted to only a few products. Recently, nanotechnology has been emerged as an active technique that has revolutionized the many sectors in an advantageous way. Elicitation using nanoparticles has been recognized as an effective strategy for enhancing the bioactive compounds of interest in plants. Nanoparticles elicit the activity of defense-related compounds through activation of the specific transcription factors involved in specialized metabolites production. This review discusses the recent progress in using nanoparticles to enhance specialized metabolite biosynthesis using hairy root culture system and the significant achievements in this area of research. Biotic and abiotic elicitors to improve the production of bioactive compounds in hairy roots, different types of nanoparticles as eliciting agents, their properties as dependent on shape, most widely used nanoparticles in plant hairy root systems are described in detail. Further challenges involved in application of nanoparticles, their toxicity in plant cells and risks associated to human health are also envisaged. No doubt, nanoparticle elicitation is a remarkable approach to obtain phytochemicals from hairy roots to be utilized in various sectors including food, medicines, cosmetics or agriculture but it is quite essential to understand the inter-relationships between the nanoparticles and the plant systems in terms of specifics such as type, dosage and time of exposure as well as other important parameters.

A simple method for creating transgenic pea hairy roots using a Japanese pea cultivar and a Japanese Rhizobium radiobacter strain.

Pea (Pisum sativum) is an agriculturally important leguminous crop cultivated worldwide. It is also the plant from which phytoalexin was isolated for the first time. Several studies have investigated gene functions using pea hairy root culture systems. However, the procedures for producing hairy roots are relatively complicated and only a few pea cultivars and Rhizobium strains have been used. In this study, we established a simple method for generating transgenic hairy roots using a pea cultivar and a Rhizobium strain available in Japan. The transformation efficiency for the transgenic hairy roots (approximately 14%) was calculated on the basis of GFP fluorescence because the binary vector used in this study carried a GFP cassette as a marker. Furthermore, we confirmed that the production of the phytoalexin (+)-pisatin was induced by a copper dichloride treatment, indicating that this system can be used to characterize the biosynthesis of (+)-pisatin, which is a compound with a unique pterocarpan structure. Interestingly, some of the hairy roots turned into crown galls during the culture period. In summary, our simple method enables the production of transgenic pea hairy roots using biological materials accessible in Japan. The generated hairy roots can be used to elucidate the molecular mechanisms underlying (+)-pisatin biosynthesis as well as hairy root/crown gall formation.

Overexpression of geraniol-10-hydroxylase improves valtrate accumulation in Valeriana jatamansi

AbstractValtrate is the major active component of iridoids in Valeriana jatamansi plants, and has been widely used for the treatment of various diseases, giving rise to rapidly increasing market demands. The gene VjG10H from V. jatamansi encoding geraniol 10-hydroxylase (G10H) was introduced into V. jatamansi hairy roots and a transgenic hairy root culture system of V. jatamansi with good valtrate production ability was successfully established for the first time. The valtrate content of VjG10H-transformed lines ranged from 8.12 to 10.77 mg/g, with an average valtrate content (9.52 mg/g) of all the four G lines being higher than the non-transgenic hairy root line. Overexpression of VjG10H significantly improved valtrate production, indicating that G10H played an important role in stimulating valtrate accumulation. MeJA treatment stimulated valtrate accumulation in all of the VjG10H overexpression recombinant lines compared to untreated cultures, indicating that methyl jasmonate treatment was another effective regulation target for metabolic engineering of valtrate biosynthesis in V. jatamansi. Thus, transgenic hairy root technology coupled with elicitor treatments may be a promising strategy to increase valtrate yield in V. jatamansi.

Effects of various Agrobacterium rhizogenes strains on hairy root induction and analyses of primary and secondary metabolites in Ocimum basilicum.

The hairy root (HR) culture system is an excellent alternative strategy to the whole plant system for producing valuable compounds. However, selection of suitable Agrobacterium strain for the successful induction of HR is an essential step for enhanced production of beneficial secondary metabolites. In this study, we examined the transformation efficiency of various A. rhizogenes strains (ATCC 13333, ATCC 15834, A4, R1000, R1200, and R1601) for transgenic HRs induction in Ocimum basilicum. Among the tested strains, the R1601 was found to be one of the most promising strain for mass production of HR in terms of transformation efficiency (94%) and the number and length of HR (8.4 ± 0.52 and 1.68 ± 0.14 cm). The HR induced by the same strain exhibited highest levels of rosmarinic acid level (62.05 ± 4.94 µg/g DW) and total phenolic content (62.3 ± 4.95 µg/g DW). A total of 55 metabolites were identified using high-performance liquid chromatography (HPLC) and gas chromatography-time-of-flight mass spectrometry (GC-TOFMS). The PCA and PLS-DA plot of the identified metabolites showed that HR induced by A4 and ATCC 15834 displayed variation in primary and secondary metabolite contents. Analysis of the metabolic pathway identified a total of 56 pathways, among which 35 were found to be impacted. A heat map and hierarchical clustering analysis indicated that HR induced by different Agrobacterium strains exhibited differential metabolites profiles. In conclusion, Agrobacterium strains R1601 is one of the best and most promising strains for inducing mass HR production and enhanced levels of secondary metabolites in O. basilicum.

Root Cultures, a Boon for the Production of Valuable Compounds: A Comparative Review.

Medicinal plants are an inevitable source of pharmaceutical drugs and most of the world population depends on these plants for health benefits. The increasing global demand for bioactive compounds from medicinal plants has posed a great threat to their existence due to overexploitation. Adventitious root and hairy root culture systems are an alternative approach to the conventional method for mass production of valuable compounds from medicinal plants owing to their rapid growth, biosynthetic and genetic stability. The main purpose of this review is to investigate the recent scientific research published worldwide on the application of adventitious and hairy root cultures to produce valuable compounds from medicinal plants. Furthermore, a comparison of adventitious root vs. hairy root cultures to produce valuable compounds has also been discussed. Various aspects such as medium composition, carbon source, pH, amount of macronutrients, optimization strategy, scale-up cultures, and use of biotic abiotic and nano-elicitors at various concentrations are the topic of discussion in this review. Several studies on adventitious and hairy root cultures of Polygonum multiflorum¸ Withania somnifera¸ Echinacea purpurea and Ajuga bracteosa have been discussed in detail which highlights the importance of elicitation strategies and bioreactor system, presenting commercial applications.

Full-Length Transcriptome Analyses of Genes Involved in Triterpenoid Saponin Biosynthesis of Psammosilene tunicoides Hairy Root Cultures With Exogenous Salicylic Acid.

Triterpenoid saponins constitute a diverse class of bioactive compounds in medicinal plants. Salicylic acid (SA) is an efficient elicitor for secondary metabolite production, but a transcriptome-wide regulatory network of SA-promoted triterpenoid saponin biosynthesis remains little understood. In the current study, we described the establishment of the hairy root culture system for Psammosilene tunicoides, a triterpenoid saponin-producing medicinal herb in China, using genetic transformation by Agrobacterium rhizogenes. Compared to controls, we found that total saponin content was dramatically increased (up to 2.49-fold) by the addition of 5 mg/L SA in hairy roots for 1 day. A combination of single-molecule real-time (SMRT) and next-generation sequencing (Illumina RNA-seq) was generated to analyze the full-length transcriptome data for P. tunicoides, as well as the transcript profiles in treated (8 and 24 h) and non-treated (0 h) groups with 5 mg/L SA in hairy roots. A total of 430,117 circular consensus sequence (CCS) reads, 16,375 unigenes and 4,678 long non-coding RNAs (lncRNAs) were obtained. The average length of unigenes (2,776 bp) was much higher in full-length transcriptome than that derived from single RNA-seq (1,457 bp). The differentially expressed genes (DEGs) were mainly enriched in the metabolic process. SA up-regulated the unigenes encoding SA-binding proteins and antioxidant enzymes in comparison with controls. Additionally, we identified 89 full-length transcripts encoding enzymes putatively involved in saponin biosynthesis. The candidate transcription factors (WRKY, NAC) and structural genes (AACT, DXS, SE, CYP72A) might be the key regulators in SA-elicited saponin accumulation. Their expression was further validated by quantitative real-time PCR (qRT-PCR). These findings preliminarily elucidate the regulatory mechanisms of SA on triterpenoid saponin biosynthesis in the transcriptomic level, laying a foundation for SA-elicited saponin augmentation in P. tunicoides.

Genetic Transformation using Agrobacterium rhizogenes for the Production of Valuable Anti-Cancer Compound, Withaferin-A from Withania somnifera (L.) Dunal

Withania somnifera (L.) Dunal is an important ayurvedic medicinal plant and its primary bioactive compound, withaferin-A possess several pharmaceutical and therapeutic values ranging from immunomodulation to anti-cancer property. For commercial production of withaferin-A, field grown plant materials have generally been used but the quality and uniformity in the levels of active constituents are highly affected by genotype and environmental conditions. Plant tissue culture methods are best alternative way to enhance commercial production of withaferin-A. In this present study, we summarize our recent findings on characterization and screening of wild accessions of W. somnifera collected from Tamil Nadu state. Further, an improved Agrobacterium rhizogenes mediated hairy root culture system was developed and this system providing an efficient tool attaining better transformation efficiency and useful for commercial in vitro production of withanolides.

Enhanced flavonoid production in hairy root cultures of Scutellaria bornmuelleri by elicitor induced over-expression of MYB7 and FNSП2 genes

For the purpose of the current study, hairy root induction in S. bornmuelleri, which is an important medicinal plant, was examined using a particular protocol. Accordingly, some factors such as four strain types of Agrobacterium rhizogenes (A4, A13, MSU440 and ATCC15834), three different explants, namely stem, petiole and leaf, two co-cultivation media, i.e. full and half-MS were studied. Besides, two inoculation methods including injection and immersion as well as three inoculation times (5, 7 and 10 min) were closely taken into account. Utilizing injection method by MSU440 strain, hairy root induction took place in stem explants, and a remarkable increase in transformation frequency (100%) was observed in half-strength MS medium. Methyl jasmonate (MeJA, 100 μM), methyl-b-cyclodextrin (b-CD, 0.7, 7 and 14 mM) and Chitosan (Chi, 50, 100 and 200 mg/l) were used either individually or in a combined way to elicitation. Based on the HPLC results, production of chrysin, wogonin and baicalein increased 9.15, 10.56 and 13.25 times after elicitation of hairy roots by MeJA + Chi. In addition, transcripts of FNSП2 and MYB7, two important genes involved in the flavonoid biosynthesis pathway, were studies. By applying Chi and MeJA + Chi elicitor, the expression of both genes increased noticeably. It can be concluded that the mentioned hairy root culture system of S. bornmuelleri can be an alternative to flavonoids production. Moreover, there is a direct and positive relationship between the expression of FNSП2 and MYB7 genes as well as the level of three flavonoids.

Hairy root culture as a valuable tool for allelopathic studies in apple.

Allelopathic plants exploit their chemical 'weapons' to prevail over the competition, suppress neighboring plants and consequently use the available resources more efficiently. However, the investigation of plant allelopathic interactions in rhizosphere is difficult to perform because of its high complexity due to interactions of biotic and abiotic factors. Thus, autonomous, aseptic root cultures of apple (Malus × domestica Borkh.) could facilitate allelopathic studies. We report on the successful genetic transformation of apple cultivars Melrose, Golden Delicious, Čadel and Gloster using Agrobacterium rhizogenes (Riker et al. 1930) Conn 1942 strain 15834 and for the first time the establishment of apple autonomous and permanent in vitro hairy root cultures that could be used as a new tool for apple allelopathic assays. Molecular characterization of transgenic hairy root lines was conducted to elucidate the possible relationship between expression of T-DNA genes and root growth characteristics that include branching. Similar content of phenolic acids (chlorogenic, caffeic, syringic, p-coumaric and ferulic), glycosilated flavonoids (rutin, quercitrin, isoquercitrin, kaempferol-3-glucoside) and flavonoid aglycones (quercetin and naringenin), and dihydrochalcone phloridzin, was detected in untransformed and transgenic apple root tissue by ultra high-performance liquid chromatography with mass spectrometry (UHPLC/(+/-)HESI-MS/MS) analyses, confirming that genetic transformation did not disturb secondary metabolite production in apple. Chlorogenic and caffeic acids and dihydrochalcones phloridzin and phloretin were detected as putative allelochemicals exuded into the growth medium in which transgenic roots were maintained for 4 weeks. Apple hairy root exudates significantly affected shoot and root development and growth of test plant Arabidopsis thaliana (L.) Heynh. seedlings after 5 or 10 days of treatment. Additionally, core cell-cycle genes CDKA1;1, CDKB2;1, CYCA3;1 and CYCB2;4 were down regulated in Arabidopsis shoots suggesting, in part, their role in inhibition of shoot growth. The present work highlighted an autonomous and permanent in vitro hairy root culture system as a valuable tool for studying allelopathic potential of apple, offering new perspective for allelopathy background elucidation in this important fruit species.

Plant-specific transcription factor LrTCP4 enhances secondary metabolite biosynthesis in Lycium ruthenicum hairy roots

Lycium ruthenicum Murr. is an important medicinal plant from traditional Chinese medicine. It contains various biologically active compounds, such as phenolics and alkaloids. These secondary metabolites are used extensively in dietary food and pharmaceutical products. However, these phenolics occur at very low concentrations in the roots, and thus, it is expensive to commercially produce them. The present study was proposed to induce a hairy root culture system for the first time in L. ruthenicum to achieve a high concentration of phenolic polyamines and other non-targeted secondary metabolites. The over-expression sequence of the TCP4 gene was retrieved from the transcriptome data of L. ruthenicum (LrTCP4-OE), and a gene construct (with pCAMBIA 1307) was integrated into the genome of L. ruthenicum by Ri-mediated genetic transformation. A total of 21 metabolites were tentatively identified by using ultrahigh-performance liquid chromatography coupled to photodiode array detector/quadrupole time-of-flight mass spectrometry (UPLC-PDA-qTOF-MS). Transgenic hairy root clones had higher relative abundances of kukoamine A and 17 other secondary metabolites than did control-type hairy roots. After 1 month, high-growth transgenic and non-transgenic hairy root lines were subjected to UPLC analysis for absolute quantification (with an authentic standard) of total kukoamine A. Transgenic hairy root lines (LrTCP4-OE) showed higher kukoamine A accumulation (0.14%) than did control hairy roots (0.11%). This enhanced productivity correlated with increased TCP4-OE activity, validating the primary role that TCP4 plays in total kukoamine A synthesis and the efficiency of non-targeted metabolomic techniques in studying plant metabolites.

Generation of α-solanine-free hairy roots of potato by CRISPR/Cas9 mediated genome editing of the St16DOX gene

Potato (Solanum tuberosum) is a major food crop, while the most tissues of potato accumulates steroidal glycoalkaloids (SGAs) α-solanine and α-chaconine. Since SGAs confer a bitter taste on human and show the toxicity against various organisms, reducing the SGA content in the tubers is requisite for potato breeding. However, generation of SGA-free potato has not been achieved yet, although silencing of several SGA biosynthetic genes led a decrease in SGAs. Here, we show that the knockout of St16DOX encoding a steroid 16α-hydroxylase in SGA biosynthesis causes the complete abolition of the SGA accumulation in potato hairy roots. Nine candidate guide RNA (gRNA) target sequences were selected from St16DOX by in silico analysis, and the two or three gRNAs were introduced into a CRISPR/Cas9 vector designated as pMgP237-2A-GFP that can express multiplex gRNAs based on the pre-tRNA processing system. To establish rapid screening of the candidate gRNAs that can efficiently mutate the St16DOX gene, we used a potato hairy root culture system for the introduction of the pMgP237 vectors. Among the transgenic hairy roots, two independent lines showed no detectable SGAs but accumulated the glycosides of 22,26-dihydroxycholesterol, which is the substrate of St16DOX. Analysis of the two lines with sequencing exhibited the mutated sequences of St16DOX with no wild-type sequences. Thus, generation of SGA-free hairy roots of tetraploid potato was achieved by the combination of the hairy root culture and the pMgP237-2A-GFP vector. This experimental system is useful to evaluate the efficacy of candidate gRNA target sequences in the short-term.

Biosynthesis and production of granatane alkaloids in hairy root cultures of Punica granatum

Punica granatum, the common pomegranate plant, contains compounds known as granatane alkaloids. These compounds possess medicinal properties that have positive effects on cardiovascular disease and reducing high blood pressure(1). Granatane alkaloids include compounds such as pelletierine, isopelletierine, pseudopelletierine, and N‐methylpelletierine. Granatane alkaloid biosynthesis is believed to occur in the root bark. In order to study the metabolism of these compounds, hairy root cultures were established by inoculating roots of seedlings with Agrobacterium rhizogenes strain MSU440(2). The bacterium harbored a pK2gw7 vector containing an eGFP (enhanced green fluorescent protein) sequence. Our ultimate goal is to use next generation sequencing on biochemically and transcriptionally active granatane producing tissues for gene discovery purposes. This relies on a confirmation of the hairy root culture as a tenable model system. In one of the hypothetical granatane producing pathways, the first five membered ring would originate from the decarboxylation of the amino acid lysine, which would form the compound cadaverine (1,5‐pentadiamine). The suspected enzyme for this step is PgLDC (Punica granatum lysine decarboxylase). We aim to isolate and characterize this enzyme with an established hairy root culture system. To date, several candidate genes have been identified.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

A comparative study of biotechnological approaches for producing valuable flavonoids in Prosopis farcta.

The callus and hairy root cultures of Prosopis farcta were established to develop effective strategies to enhance its valuable and medicinally important flavonoid compounds. For callus induction, the hypocotyl, cotyledon and shoot explants were subjected to different plant hormones, naphthalene acetic acid (NAA), benzylaminopurine (BAP), kinetin and dichlorophenoxyacetic acid (2,4-D). Greater callus induction was obtained from hypocotyl explants on MS medium containing 3.0mgL-1 NAA+2.0mgL-1 BAP. With the addition of 0.5mgL-1 asparagine to this medium, the maximum callus growth was achieved. Hairy root culture of P. farcta was performed using transformation of different explants with strains of Agrobacterium rhizogenes LBA9404, A4, AR15834. The AR15834 strain was more effective for hairy root induction where it caused hairy root formation on 59% of the infected cotyledon explants. We compared profiles of flavonoids isolated from seedling roots, hairy roots, and callus cultures of P. farcta. The colorimetric analysis showed that the content of total flavonoids of hairy roots was 1.54 and 2.52 times higher than in seedling roots and callus, respectively. The presence of flavonoids was verified by LC/MS in positive ion mode. The results showed that flavonoid composition was different in the roots and callus. Naringenin was the major constituent in callus, whereas resveratrol, quercetin and myricetin were the most abundant compounds found in hairy roots. The main objective of this research was to establish hairy roots in P. farcta to synthesize flavonoids at levels comparable to in vitro-grown roots. The present study also opens up a way to further improve the production of pharmaceutically valuable flavonoids and to produce desired metabolites using the hairy root culture system.

Enhanced production of pharmaceutically important isoflavones from hairy root rhizoclones of Trifolium pratense L.

These studies report the development of an efficient technique for large-scale cultivation of fast-growing hairy root culture systems for production of bioactive isoflavones. Trifolium pratense L. is an important source of pharmaceutically important isoflavones with immense health care applications. Trifolium pratense was transformed using different strains of Agrobacterium rhizogenes for hairy root induction and establishment of hairy root rhizoclones. Selected fast-growing rhizoclones of T. pratense were evaluated for their growth and isoflavone production. This study is the first report of stable production of isoflavones through successive culture passages from transformed hairy-root rhizoclones of T. pratense. One of the fast-growing hairy-root rhizoclones 2364A displayed significantly higher accumulation of all four pharmaceutically important isoflavones, 8.56 mg (gdw)−1 of daidzein, 2.45 mg (gdw)−1 of genistein, 15.23 mg (gdw)−1 of formononetin, and 1.10 mg (gdw)−1 of biochanin A, compared to other rhizoclones.

Biologically active recombinant human erythropoietin expressed in hairy root cultures and regenerated plantlets of Nicotiana tabacum L.

Hairy root culture is a potential alternative to conventional mammalian cell culture to produce recombinant proteins due to its ease in protein recovery, low costs and absence of potentially human pathogenic contaminants. The current study focussed to develop a new platform of a hairy root culture system from Nicotiana tabacum for the production of recombinant human EPO (rhEPO), which is regularly produced in mammalian cells. The human EPO construct was amplified with C-terminal hexahistidine tag from a cDNA of Caco-2 cells. Two versions of rhEPO clones, with or without the N-terminal calreticulin (cal) fusion sequence, were produced by cloning the amplified construct into gateway binary vector pK7WG2D. Following Agrobacterium rhizogenes mediated transformation of tobacco explants; integration and expression of constructs in hairy roots were confirmed by several tests at DNA, RNA and protein levels. The amount of intracellular rhEPO from hairy root cultures with cal signal peptide was measured up to 66.75 ng g-1 of total soluble protein. The presence of the ER signal peptide (cal) was essential for the secretion of rhEPO into the spent medium; no protein was detected from hairy root cultures without ER signal peptide. The addition of polyvinylpyrrolidone enhanced the stabilization of secreted rhEPO leading to a 5.6 fold increase to a maximum concentration of 185.48 pg rhEPOHR g-1 FW hairy root cultures. The rhizo-secreted rhEPO was separated by HPLC and its biological activity was confirmed by testing distinct parameters for proliferation and survival in retinal pigment epithelial cells (ARPE). In addition, the rhEPO was detected to an amount 14.8 ng g-1 of total soluble leaf protein in transgenic T0 generation plantlets regenerated from hairy root cultures with cal signal peptide.

Efficient Agrobacterium Rhizogenes Mediated Hairy Root Culture System for Withanolide Production from Withania Somnifera (L.) Dunal

Withania somnifera (L.) Dunal is an important medicinal plant with many pharmaceutical and therapeutic uses ranging from immunomodulation to anticancer property. Field cultivation of W. somnifera is time consuming and it not able to meet current global demands. For commercial production of withanolides, field grown plant materials have generally been used but the quality and uniformity in the levels of active constituents are highly affected by genotype and environmental conditions. In vitro culture methods are the best alternative and have possible ways to enhance commercial production of withanolides. In this review, we summarize our recent findings on characterization and screening of wild accessions of W. somnifera collected from Tamil Nadu state. Further, an improved Agrobacterium rhizogenes mediated hairy root culture system was developed and this system providing an efficient tool attaining better transformation efficiency and useful for commercial in vitro production of withanolides.

Addressing Challenges to Enhance the Bioactives of Withania somnifera through Organ, Tissue, and Cell Culture Based Approaches.

Withania somnifera is a highly valued medicinal plant in traditional home medicine and is known for a wide range of bioactivities. Its commercial cultivation is adversely affected by poor seed viability and germination. Infestation by various pests and pathogens, survival under unfavourable environmental conditions, narrow genetic base, and meager information regarding biosynthesis of secondary metabolites are some of the other existing challenges in the crop. Biotechnological interventions through organ, tissue, and cell culture provide promising options for addressing some of these issues. In vitro propagation facilitates conservation and sustainable utilization of the existing germplasms and broadening the genetic base. It would also provide means for efficient and rapid mass propagation of elite chemotypes and generating uniform plant material round the year for experimentation and industrial applications. The potential of in vitro cell/organ cultures for the production of therapeutically valuable compounds and their large-scale production in bioreactors has received significant attention in recent years. In vitro culture system further provides distinct advantage for studying various cellular and molecular processes leading to secondary metabolite accumulation and their regulation. Engineering plants through genetic transformation and development of hairy root culture system are powerful strategies for modulation of secondary metabolites. The present review highlights the developments and sketches current scenario in this field.

13C NMR and LC-MS Profiling of Stilbenes from Elicited Grapevine Hairy Root Cultures.

Resveratrol and related oligostilbenes are defense molecules produced by grapevine in response to stresses including various elicitors or signal molecules. Together with their prominent role in planta, these compounds have been the center of much attention in recent decades due to their pharmacological properties. The cost-effective production of resveratrol derivatives such as viniferins or more structurally complex stilbene oligomers remains a challenging task. In this study, the chemical diversity of stilbenes produced by Vitis vinifera Pinot Noir hairy roots was investigated after elicitation for 4 days with a mixture of methyl jasmonate (100 μM) and cyclodextrins (50 mM). Two crude extracts obtained from the culture medium and from the hairy roots were fractionated by centrifugal partition chromatography. The fractions were chemically investigated by two complementary identification approaches involving a 13C NMR-based dereplication method and liquid chromatography coupled to mass spectrometry (LC-MS). In total, groups of 21 and 18 molecules, including flavonoids and stilbenes, were detected in the culture medium and root extracts, respectively. These included resveratrol monomers, dimers, trimers, and a tetramer, thus highlighting the ability of elicited hairy root culture systems to synthesize a wide diversity of secondary metabolites of pharmaceutical significance. The main compounds were unambiguously identified as trans-resveratrol, ε-viniferin, trans-piceatannol, pallidol, scirpusin A, eriodictyol, naringenin, vitisin B, and maackin.

Prunella vulgaris L. hairy roots: Culture, growth, and elicitation by ethephon and salicylic acid

Hairy roots have gained popularity as an alternative source for secondary metabolites not only because of their biosynthetic and bioactive potentials, but also because of studies of experimental systems aiming to understand secondary metabolic pathways. While many secondary metabolites have been identified in Prunella vulgaris, a comprehensive understanding of the metabolic pathways has remained elusive. Here, Agrobacterium rhizogenes (ATCC15834)‐mediated hairy root induction of P. vulgaris resulted in 15–30 times more rosmarinic acid (RA) than in the intact plant. After the growth and RA biosynthesis of P. vulgaris, hairy roots were investigated over a 1‐month period, we studied elicitation by ethephon (Eth) and salicylic acid (SA) and found 200 μg L−1 Eth and 6.9 mg L−1 SA exhibited the optimum induction efficiency. These concentrations were further selected for a time‐course assay of hairy root cultures to assess exposure to these compounds over 8 days. During that period, RA reached maximum accumulations of 1.66‐fold 8 days after Eth elicitation and 1.48‐fold 2 days post‐SA addition. We also examined the transcripts of genes involved in the RA biosynthesis pathway, and found the expression of tyrosine aminotransferase (TAT) and phenylalanine ammonia‐lyase (PAL) were increased by Eth, and SA heightened the expression of TAT, 4‐hydroxyphenylpyruvate reductase (HPPR), PAL, 4‐coumaric acid CoA‐ligase 1 (4CL1), and cytochrome P450‐dependent monooxygenase (CYP). Altogether, our results indicate that the convenient hairy root culture system of P. vulgaris can provide an alternate source for RA production and would be conducive for the engineering of secondary metabolic pathways in P. vulgaris.