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The Himalayan Yew or Taxus baccata sub sp. wallichiana is the native of the Temperate belt of Himalayas known widely for its ethnomedical properties and especially for the production of an anti-cancerous compound Taxol but since the plant is an understory and in low population size, the production of Taxol in nature is very poor so it cannot meet the high demand of Taxol for medical use. Hairy root culture is known for its genetic and physiochemical stability and increased production of secondary metabolites as compared to cell suspension culture and with the use of low frequency sonication for 1–2 min, the rate of transformation can be increased thereby induing more hairy roots. In our study, when the combination of low frequency sonication for production of hairy roots was used it helped in development of the hairy root culture of Taxus baccata sub sp. wallichiana as an alternative for increased Taxol production. It was seen that the hairy root induction from the explants emerged when an effective co– infection method in presence of acetosyringone with sonication for 60 s was done. The increase in hairy root biomass was further done for 16 weeks in root induction media that was without acetosyringone. The methanolic extract of the biomass when quantified for total Taxol content produced around 12 mg of Taxol per gram of hairy root biomass of Taxus baccata sub sp. wallichiana which is 79% more than the natural production of 0.15 mg per gram of bark stating a possible alternative for Taxol production commercially in a large amount.

Bacoside is a phytocompound found in Bacopa monnieri (BM) which is very useful to treat various disorders like Alzheimer’s, Parkinson’s, cancer, ulcer, bronchitis, asthma, and a number of other diseases. It is a tetracyclic triterpenoid saponins-dammarane; various types of bacosides are present in the plant BM which includes bacoside A, bacoside B, bacopaside and bacopasaponin. Due to its pharmaceutical importance, there is an increase in demand for this plant which is leading towards the extinction of the plant. In natural conditions production of bacoside is low due to various environmental factors and species variations. Production of bacoside is time-consuming and not feasible, when exploiting conventional methods. Production of bacoside can be enhanced by using biotechnological approaches such as cell suspension culture, shoot culture, root culture and hairy root culture. By providing essential media, nutrients and optimum growth conditions production of bacosides can be improved. Bioreactors can also be used for the large-scale production of bacosides as they provide a suitable environment for growth of the plant. Therefore, in this review chemistry of bacosides, biosynthesis, pharmaceutical applications and production enhancement strategies have been discussed.

Demand for naturally-derived compounds is growing steadily, owing to a broad spectrum of medicinal benefits posed by them. Triterpenoid centellosides, obtained from Centella asiatica, is being utilized as an important component in a variety of products such as pharmaceutics. Centellosides possess various therapeutic properties like antidiabetic, anti-inflammatory, antioxidant, anticancer, wound healing, neuroprotective properties, etc. However, C. asiatica is reportedly undergoing rapid depletion and is endangered due to the increasing demand of this plant for its secondary metabolites. Apart from this cultivation of C. asiatica needs a long time period, along with extensive labour for controlling the quality despite the unfavourable environmental conditions. Therefore, a substitute method is required for its cultivation and improved production of centellosides. For biosynthesis of centellosides at higher levels, various biotechnological methods have now been established, such as shoot culture, callus and cell suspension culture and hairy root culture. Further, many more strategies like use of elicitors and precursors for enhanced production as well as bioreactors for commercial scaling have also been incorporated. This review thus provides an understanding of biological applications of centellosides, biotechnological approaches as well as biotechnological improvements for its increased production. Information about various biotechnological approaches has also been collected after an extensive literature study. Centellosides are important bioactive compounds from Centella asiatica which possess various therapeutic properties like antidiabetic, anti-inflammatory, antioxidant, anticancer, wound healing, neuroprotective properties, etc. For biosynthesis of centellosides at higher levels, various biotechnological methods have been established, such as shoot culture, callus and cell suspension culture, and hairy root culture. This review provides an understanding of biological applications of centellosides, biotechnological approaches as well as biotechnological improvements for its increased production.

Demand for compounds from natural sources are increasing day by day. Ginsenosides are secondary metabolites, belonging to triterpenoid saponin groups which are present in Panax species. Ginsenosides possess wide range of therapeutic activities which includes anti-aging, anti-oxidative, anti-cancerous, etc. Cultivation of plants containing ginsenosides requires a long duration of time, ranging from five to seven years, along with extensive efforts for controlling the quality despite of the unfavorable environmental conditions. Isolation and production of ginsenosides is also time-consuming and not feasible, when exploiting the conventional methods. Therefore, an alternative method is required for its enhanced production. Biotechnological methods such as shoot culture, cell suspension culture, root culture and hairy root culture are largely being executed for enhanced production of ginsenosides. Bioreactors are also utilized for scaling up the production of ginsenosides. Therefore, this review provides a comprehensive information about pharmaceutical applications of ginsenosides, different biotechnological approaches (shoot, root, hairy root and callus/cell suspension culture) that are used for the production of ginsenosides and scale up strategies for large scale production. Information about different in vitro systems has also been compiled after a panoramic literature survey and last twenty years data has been taken into consideration for this review.

Peanut (Arachis hypogaea L.) hairy roots are a potential tool for stilbene compound production. This study focuses on the most efficient elicitation strategy for inducing stilbene compound production, especially for the prenylated forms of trans-arachidin-1 and trans-arachidin-3. To the best of our knowledge, this is the first report of simultaneous treatment involving chitosan (CHT), methyl jasmonate (MeJA), and cyclodextrin (CD); CHT+MeJA+CD in peanut hairy roots to induce substantial amounts of trans-arachidin-1 and trans-arachidin-3 at 72 h of the elicitation period. The results demonstrate the highest amounts of trans-arachidin-1 and trans-arachidin-3, with 684.30 ± 183.85 and 543.94 ± 171.17 mg/g dry weight of hairy roots, respectively. The highest antioxidant capacity determined by ABTS and FRAP assays was 188.95 ± 0.43 µmol Trolox/g dry weight of hairy roots and 24.61 ± 0.50 mg ascorbic acid/g dry weight of hairy roots, respectively, while the highest phenolic content was also detected in this crude extract, with 8.67 ± 0.11 mg gallic acid/g dry weight of hairy roots. The antioxidant and pro-oxidant activity of the CHT+MeJA+CD crude extract in the protection and damage of DNA is of great interest and can have major positive impacts on health promotion and disease prevention.

Celastrus paniculatus Willd., an important medicinal plant for the treatment of neurodegenerative diseases is at risk of extinction due to destructive harvesting by pharmaceutical industries in its different habitats. This study aims to determine the impact of silver nanoparticles on the enhancement of therapeutic Celastrol content in adventitious and hairy root culture. MS medium supplemented with 0.3 mg L−1 Indole Acetic Acidwas found suitable for adventitious root induction (69%). Hairy roots were successfully induced by co-cultivation of leaf explants with Agrobacterium rhizogenes strain MTCC532 supplemented with acetosyringone (100 µM) to the half-strength medium. Transformation confirmed by PCR analysis.10 µg mL−1 of AgNPs with 48 h exposure time resulted in enhanced biomass accumulation in adventitious roots and in hairy roots. HPLC analysis confirmed that Celastrol content increased 2.24-fold and 1.87-fold higher in hairy root and adventitious roots respectively.

Sahendian savory (Satureja sahendica Bornm) is a medicinal plant species which produces valuable secondary metabolites including thymol, p-cymene and γ-terpinene. In this study, optimization of hairy roots culture in S. sahendica using Agrobacterium rhizogenes was efficiently established. High frequency of genetic transformation was obtained using node and internode explants and three bacterial strains (ATCC 15834, A4, and LBA 9402). However, leaf explants showed no susceptibility to induce hairy roots with the A. rhizogenes strains. The node explants and LBA 9402 strain were the best combination for root induction. Infection time of 20 min caused greater transformation occurrence compared to 10 and 30 min, respectively. Node explants infected with LBA 9402 were found to produce more transformants upon co-cultivation for 2 days. The maximum induction of hairy roots was obtained from bacterial infection at a density of OD600 = 0.8. Polymerase chain reaction by gene-specific primers from rol B gene confirmed the positive transformation events. S. sahendica efficiently provided and produced a large number of hairy roots which could be used for the production of secondary metabolites or other molecular and biotechnological applications. Production of thymol an important secondary metabolite of S. sahendica from hairy root cultures was confirmed by TLC and GC analysis. Elicitor-treated hairy root cultures with methyl jasmonate and ammonium nitrate produced more amount of thymol compared to untreated roots. These results demonstrate that secondary metabolite production such as thymol in hairy root culture of Sahendian savory could be used to increase low secondary metabolite production in this valuable medicinal plant.

Curcumin has ignited global interest as an elite drugable molecule, owing to its time-honoured pharmacological activities against diverse human ailments. Limited natural accessibility and poor oral bioavailability caused major hurdles in the curcumin-based drug development process. We report the first successful testimony of curcumin and its glucoside synthesis in Atropa belladonna hairy roots (HR) through metabolic engineering. Re-routing the inherent biosynthetic precursors of the phenylpropanoid pathway of A. belladonna by heterologous expression of key curcumin biosynthetic pathway genes (i.e., Diketide-CoA synthase-DCS and Curcumin synthase-CURS3) and glucosyltransferase gene (CaUGT2) resulted in the production of curcumin and its glucoside in HR clones. Under shake-flask cultivation, the PGD2-HR1clone bearing DCS/ CURS3 genes showed the maximum curcumin yield (180.62 ± 4.7 μg/g DW), while the highest content of curcumin monoglucoside (32.63 ± 2.27 μg/g DW) along with curcumin (67.89 ± 2.56 μg/g DW) were noted in the PGD3-HR3 clone co-expressing DCS/CURS3 and CaUGT2 genes. Bioreactor up-scaling showed yield improvements in the PGD2-HR1 (2.3 fold curcumin) and the PGD3-HR3 clone (0.9 and 1.65 folds of curcumin-monoglucoside and curcumin respectively). These findings proved the advantageous use of HR cultures as the production source for curcumin and its glucoside, which remained unexplored so far.

Plants encounter diverse stressors simultaneously with changing environmental factors. The combined effect of different types of stresses can have a wide range of effects on plants. The present study demonstrated that various stress factors such as the combination of chemical elicitors, namely paraquat (PQ), methyl jasmonate (MeJA) and methyl-β-cyclodextrin (CD), light exposure versus darkness, and mechanical shearing stress affected the defence response in peanut hairy root culture. The antioxidant activities were dramatically increased at all time points after hairy roots were subjected to elicitation with PQ + MeJA + CD under root cutting in both light and dark conditions. The stilbene compounds were highly increased in the culture medium after elicitor treatment of uncut hairy roots under dark conditions. In contrast to the high stilbene contents detected in culture medium under dark conditions, the transcription of the stilbene biosynthesis genes PAL, RS and RS3 was enhanced by the effect of light in uncut hairy root tissues. The antioxidant enzyme genes APX, GPX and CuZn-SOD of uncut and cut hairy roots were more highly expressed in light conditions than in dark conditions. The pathogenesis-related protein (PR)-encoding genes chitinase, PR4A, PR5 and PR10 of uncut hairy roots were highly expressed in response to light conditions compared to dark conditions at all time points. Recent evidence of the production of antioxidant stilbene compounds and defence response genes has implicated plant protective functions through defence responses under different stress challenges. Plant responses might therefore be regulated by the coordination of different signal responses through dynamic pathways.

Papaver armeniacum hairy roots were induced by four Rhizobium rhizogenes strains on three explants (shoot, root, and hypocotyl). Also, the effects of two concentrations (100 and 200 μM) of methyl jasmonate (MJ) and salicylic acid (SA) were assessed on productions of papaverine, noscapine, thebaine, morphine, and codeine and expression of some related genes (TYDC, DBOX, BBE, SalAT, T6ODM, and COR) in P. armeniacum L. hairy root culture at 24 and 48 h after elicitation. R. rhizogenes strain C58C1 induced the highest hairy root rate on hypocotyl explant. Application of 100 μM MJ resulted in the highest contents of thebaine, codeine, and morphine by enhancing the expression of SalAT, COR, and T6ODM genes, respectively, while application of 100 μM SA resulted in the highest contents of papaverine and noscapine by upregulating DBOX and BBE genes, respectively. 100 μM MJ can be used as an effective elicitor in P. armeniacum hairy root culture to increase studied morphinan alkaloids. Also, SA can be suggested for enhancing papaverine and noscapine contents in P. armeniacum hairy root culture. It may be due to that there is a SA- and MJ-signaling crosstalk, which results in reciprocal antagonism between SA and MJ signaling pathways. The effects of MJ and SA elicitors on benzylisoquinoline alkaloids (BIAs) production were level-dependent.

This study evaluated the potential of dwarf morning-glory Convolvulus tricolor (Convolvulaceae) plants and their hairy roots induced by Agrobacterium rhizogenes for rhizoextraction of heavy metals ions from the liquid medium under aseptic growth conditions. Both the young C. tricolor plants and the generated hairy root culture efficiently removed Cr6+, Ni2+, and Pb2+ ions from the liquid cultivation medium. As determined by atomic absorption spectroscopy, the hairy roots demonstrated a high level of heavy metal ions accumulation (μg/g dry weight): 3942 ± 210 of chromium, 1529 ± 312 of nickel, and 2613 ± 373 of lead. These data show that the hairy roots of morning glory might be of interest for some phytoremediation strategies due to their high bioaccumulation abilities. The comparison of bioaccumulation potential of established hairy roots and young C. tricolor plants give grounds to suppose that roots of C. tricolor play an active role in the absorption of Cr6+, Ni2+, and Pb2+ from liquid media, whereas the aboveground part rather serves as a storage for the accumulated metal ions.

Among various in vitro plant culture systems, hairy root systems seem to be one of the most appealing methods of recombinant protein production due to their advantages in combining both whole-plant cultivation and suspension cell culture platform. This is a report on production and secretion of a recombinant pharmaceutically active protein from hairy roots cultures of Withania somnifera to improve the economic potential of this plant for the production pharmaceutical compounds. In this study, we selected and synthesized a codon-optimized globular adiponectin (gAd) gene with a calreticulin signal peptide and cloned the sequence into a plant expression binary vector containing a nptII gene as a selectable marker gene. The transgenic hairy roots were produced by Agrobacterium rhizogenes-mediated transformation protocol developed by our group. Among ten established nptII positive hairy roots lines, six colons significantly accumulated gAd protein in the biomass and extracellular medium. The presence of gAd was confirmed by western blot analysis of root extracts. The maximum level of hairy root biomass, growth rate (GR), intra- and extracellular gAd expressions were obtained after 25 to 26 days of culture on MS medium. The maximum level of intra- and extracellular gAd proteins were found to be 15.19 µg/gFW and 215.7 µg/L, respectively, which resulted in a significant decrease in the amount of intra- and extracellular withanolide A and withaferin A production. The addition of PVP, KNO3 and NaCl significantly increased the level of extracellular gAd by approximately 13 folds. This improvement could significantly increase the amount of intra- and extracellular withanolide A and withaferin A production, too. The recombinant gAd produced from W. somnifera is functional as proved by induction the phosphorylation of ACC in C2C12 muscle cells, as its functional amount was 5.1-fold more than gAd produced from E. coli and 45% lower than CHO cells.

Hairy root cultures are often used to produce valuable metabolites. They are grown on sucrose-rich medium, which is highly susceptible to contamination. Trapa natans is a unique plant with photosynthesizing roots. It is a promising object to obtain photoautotrophic hairy root culture. Protocols for transformation of this species are yet unknown. We report that hairy roots can be induced in aquarium and in vitro cultures of T. natans by agrobacterium-mediated and biolistic transformation. 64 roots were induced by Agrobacterium rhizogenes strain 15834, two roots were obtained using strain K599. Strain A4 was not effective. Biolistics with either amplicons of rol genes and 1301 pCAMBIA plasmid carrying rol genes resulted in the formation of six roots. All these roots contained chloroplasts. This achievement opens a prospect for genetic transformation of T. natans and use of its green photosynthesizing hairy root cultures in production of bioactive substances and in phytoremediation. Protocols for the Agrobacterium-mediated and biolistic transformation of Trapa natans to induce hairy roots have been developed for the first time. All induced roots contained chloroplasts.

The dried root of Astragalus membranaceus is a well-known herbal medicine, and it is useful in treating chronic diseases and weakness, as well as for improving overall health and vitality. Astragalosides, which are root quality indicators of A. membranaceus, are natural triterpenoid saponins that are used in the treatment of diabetes and cardiovascular diseases. Currently, there is an urgent need to improve their production because of their low quantity in plants and the difficulty of chemical synthesis. In this study, yeast extract was added to facilitate elicitation in Agrobacterium-mediated hairy root cultures, thereby enhancing astragaloside production in A. membranaceus. Results showed that yeast extract could stimulate astragaloside content effectively in the hairy roots of A. membranaceus. Moreover, astragaloside accumulation was positively correlated with the upregulation of mevalonate biosynthetic gene expression in the presence of yeast extract. Our study demonstrated that pretreatment with yeast extract (3.65 mM) for 72 h serves as an effective strategy to enhance astragaloside levels in A. membranaceus hairy root cultures. Thus, these optimal conditions can provide valuable information for the improvement of astragaloside industrial production in A. membranaceus.

Cyst nematodes (Globodera spp. and Heterodera spp.) are highly evolved sedentary endoparasites that are considered as harmful pests worldwide. The hatching of the dormant eggs of cyst nematodes occurs in response to hatching factors (HFs), which are compounds that are secreted from the roots of host plants. Solanoeclepin A (SEA), a triterpene compound, has been isolated as HF for potato cyst nematode (PCN) eggs, whereas other compounds, such as steroidal glycoalkaloids (SGAs), are also known to show weak hatching stimulation (HS) activity. However, the structures of both compounds are different and the HF-mediated hatching mechanism is still largely unknown. In the present study, we observed specific hatching of PCN eggs stimulated by the hairy root culture media of potato and tomato, revealing the biosynthesis and secretion of HFs. SGAs, such as α-solanine, α-chaconine, and α-tomatine, showed significant HS activity, despite being remarkably less activities than that of SEA. Then, we evaluated the contribution of SGAs on the HS activities of the hairy root culture media. The estimated SGAs content in the hairy root culture media were low and nonconcordant with the HS activity of those, suggesting that the HS activity of SGAs did not contribute much. The analysis of structure-activity relationship revealed that the structural requirements of the HS activity of SGAs are dependent on the sugar moieties attached at the C3-hydoroxyl group and the alkaloid property of their aglycones. The stereochemistry in the EF rings of their aglycone also affected the strength of the HS activity.

Salvia viridis transformed root culture was studied using MS, WP or B5 media (full or half-strength macro- and microelements: 1/2MS, 1/2WP, 1/2B5) under varying light conditions to optimize growth and polyphenolic compound production. The highest levels of root growth (154.6 g/l and 13.6 g/l, respectively, fresh and dry weight) were observed for hairy roots grown in WP medium in the dark. UPLC analysis found the polyphenolic acid content in plant material to range from 20.7 to 45.9 mg/g dry weight (DW) depending on the conditions. The highest metabolite content was observed in roots grown in B5 medium under photoperiods. A time-course study of the hairy roots cultured in WP medium in the dark examined the effect of culture period on biomass and polyphenolic compound yield every 5 days over a 60-day period. More than 1 g of dry weight per flask and 47.8 mg of metabolite per g DW were produced after 30 days, at the beginning of the stationary phase. However, maximum production was only observed for four of the 10 detected compounds during this growth phase, between days 30 and 40; one of which was rosmarinic acid: the predominant compound in all extracts. Under optimal conditions, production was almost 10 times higher than that observed in the roots of field growing plants, which suggests that the selected optimized transformed root culture of S. viridis may be a good approach for valuable polyphenol production. The present study reports the influence of medium type, light condition and culture period on the accumulation of phenolic acids in hairy roots of S. viridis.

Plants belonging to the genus Artemisia L. have been used for medicinal purposes since ancient times. These aromatic plants produce and accumulate a wide range of potent secondary metabolites, many of which have shown antioxidant, antiparasitic, antimicrobial, anti-inflammatory, and even anticancer activities. Enhanced biosynthesis of these compounds is a prerequisite for comprehensive studies of their therapeutic properties and cost-efficient use. Transformation of plants with Agrobacterium rhizogenes native root locus (rol) genes is a promising approach to increase the biosynthesis of plant secondary metabolites. The aim of the present study was to evaluate the effects of A. rhizogenes-mediated transformation on the flavonoid contents in hairy roots of medicinal herb A. tilesii Ledeb. Transgenic A. tilesii hairy root lines were analyzed for stable integration of the rolB and rolC transgenes into the plant genome, total flavonoid contents, antioxidant activities of extracts, and the spatiotemporal expression of two flavonoid biosynthetic genes, phenylalanine ammonialyase (PAL) and chalcone synthase (CHS). The flavonoid contents of A. tilesii directly correlated with the antiradical activity and reducing power of their respective lines, with the greatest antioxidant activity found in the plants with the highest level of total flavonoids. Furthermore, all hairy root lines demonstrated altered expression of plant native PAL and CHS genes. Most importantly, A. rhizogenes-mediated transformation enhanced the biosynthesis of natural antioxidants in A. tilesii, producing almost twice the amount of flavonoids than controls. These findings provide an opportunity for the identification of the bioactive molecules in A. tilesii extracts and their potential health benefits.

Atropa belladonna L. is an important medicinal plant due to the biosynthesis of hyoscyamine and scopolamine; two important tropane alkaloids, mostly well known for their anticholinergic properties acting on the parasympathetic nervous system. This study aimed to analyze the effect of methyl jasmonate (MJ), at different concentrations, on hyoscyamine and scopolamine production from in vitro hairy root culture and propagated plantlets of A. belladonna L. Gene expression analysis of two key enzymes PMT and H6H responsible for the biosynthesis of hyoscyamine and scopolamine, respectively, was also carried out under MJ application. MJ decreased the growth parameters in both plantlets and hairy root cultures. In shoots of the plantlets, MJ increased significantly the hyoscyamine and scopolamine contents to the highest value of 3.32 mg g−1 DW and 0.5 mg g−1 DW, respectively. Moreover, MJ up-regulated pmt2 and h6h gene expression in root of the plantlets, which is the site of biosynthesis of hyoscyamine and scopolamine, while the accumulation occurs in the aerial parts. In hairy root culture, the hyoscyamine content increased as the MJ concentration increased, and reached its highest level of 2.1 mg g−1 DW at 0.04 mM MJ, showing the same pattern as for pmt2 gene expression. Nevertheless, the scopolamine content decreased by increasing the MJ concentration as it also showed a reverse pattern compared to the h6h gene expression, presumably due to the negative effect of hyoscyamine accumulation on H6H enzyme activity.

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.

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.

Hazelnut (Corylus avellana L.), contains a valuable medicinal substance known as Paclitaxel®, which is one of the most effective anticancer drugs. The original plants produce negligible amount of paclitaxel; therefore, tissue culture techniques, especially hairy root culture, could be one of the most practical methods to enhance the amount of paclitaxel. The main goal of this study was to assess the induction of hairy roots in C. avellana. The effects of different strains of Agrobacterium rhizogenes including c58c1pRiA4, K599, and 15834, and six culture media, MS (Murashige and Skoog), half-strength MS, quarter-strength MS, WPM (woody plant media), half-strength WPM, and quarter-strength WPM, were evaluated. The results showed that the maximum amounts of the rooted explants were obtained with c58c1pRiA4 strain in quarter-strength WPM medium. The investigations of explant type (leafstalk, petiole, lamina, and stem) and different propagation media (quarter-strength WPM, half-strength MS, and half-strength SH ((Schenk and Hildebrandt) medium) showed that the leafstalk was the most optimal explant for hairy root induction, and half-strength SH was the best culture medium for growth of the hairy roots in liquid medium. HPLC analyses confirmed the presence of paclitaxel (3.2 μg g−1 (DW)) in hairy root extracts.

The production of biofilm inhibiting antimicrobial agents of plant origin is gaining significant importance in recent years. The inhibition of biofilm by natural agents is considered safe and provides greater scope for alternative antimicrobial strategies. The hairy root culture extracts established by infecting leaf explants with Agrobacterium rhizogenes R1000 and A4 strains in Raphanus raphanistrum L. subsp. sativus (L.) Domin (syn. R. sativus L.) have been studied in this regard. The total flavonoids and phenolics contents were estimated and their influence on the antioxidant and antibiofilm activities on the hairy root culture extracts were investigated. The R1000 strain produced 90.0% of hairy roots while the A4 strain produced 81.0% hairy roots in Murashige and Skoog (hormone-free ½ MS) medium containing 75 µM of acetosyringone within three days. The hormone-free ½ MS medium with 116.8 mM sucrose concentration produced highest biomass accumulation (14.11 g/L Fresh Weight (FW); 1.78 g/L Dry Weight (DW); Growth Index (GI) 7.06) after 30 days of culture compared to MS, B5 (Gamborg B5) and ½ B5 media. The integration of T-DNA of the Ri plasmid was confirmed by Polymerase Chain Reaction (PCR) analysis based on the amplification of rolB and rolC genes. The R1000 induced hairy roots extract (RHRE) produced high amount of phenolics (52.0 mg/g DW) and flavonoids (67.0 mg/g of DW). The quercetin content of RHRE (488.4 mg/g of DW) was determined by RP-HPLC and LC–MS/MS. The RHRE exhibited 92% inhibition against stable 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical and in ferric reducing antioxidant power (FRAP) assay the absorbance was 3.62 at 593 nm. The RHRE also exhibited significant antimicrobial activity against Gram-negative and Gram-positive bacteria, particularly against MDR bacterium Pseudomonas aeruginosa, by inhibiting biofilm formation and EPS production in the bacterium. Our findings indicate the industrial applications of RHRE as potent antibiofilm agent probably due to the enriched quercetin content. The RHRE could also be used to prevent food contamination and spoilage in industrial environment. Factors affecting hairy root production have been optimized in Raphnus sativus (L.). Agrobacterium rhizogenes R1000 displayed better results compared to A4. Optimum flavonoid production obtained in ½-strength MS medium and 4% sucrose. R1000 hairy roots displayed higher antimicrobial, antibiofilm and antioxidant activity. The present study is a novel approach to improve quercetin content and can be used in food industry.

In this study, the effects of methyl jasmonate (MeJA) on the phytomass and triterpenoid production of diploid and tetraploid Centella asiatica hairy roots were investigated. Hairy root cultures were obtained from diploid and induced tetraploid plants of C. asiatica infected by Agrobacterium rhizogenes strain ATCC 43057. MeJA triggered triterpenoid production in both ploidy hairy roots, whereas triterpenoids were not produced in the untreated hairy roots. Among the treatments, the 50 µM MeJA treatment yielded the maximum triterpenoid production in diploid hairy roots of 27.25 ± 0.27 µg/mg Dry weight (DW) total triterpenoid at day 21. For the tetraploid hairy root cultures, the 28th-day hairy root culture produced a maximum amount of triterpenoids of 16.29 ± 6.32 µg/mg DW in response to the 50 µM MeJA treatment, whereas the 100 µM MeJA treatment produced a similar triterpenoid amount (16.31 ± 9.24 µg/mg DW) at day 14. Moreover, in response to 50 µM MeJA, we obtained different ratios of aglycone to glycoside, i.e., 1:7 and 1:2, between the diploid and tetraploid hairy root cultures. Asiaticoside was the dominant phytochemical, followed by asiatic acid and madecassic acid. This study provides valuable information for producing triterpenoids for C. asiatica commercial products and preparations by using hairy root cultures.

The hairy root cultures are promising sources of secondary metabolites of plants, including rare and endangered species. They possess genetic and biochemical stability, unlimited growth rate in free-hormone medium, short doubling times, high biosynthetic activity and ecological purity of plant raw materials. The hairy root cultures of Tagetes patula L. can be used to produce biologically active substances with biocidal activity. The study aimed to determine the virulent strain of Agrobacterium rhizogenes and the most effective period of co-cultivation of T. patula leaf explants with an agrobacterium to induce actively growing hairy root cultures. We used 3 strains (A-4b, 8196RT and 15834). The time of infection ranged from 3 to 33 hours in increments of 3 hours. We found that 24 h is the best time of infection to induce hairy roots with the highest transformation efficiency (92%). The wild strain A. rhizogenes 15834 turned out to be the most virulent when infected leaf explants of spreading marigold. This strain provided the maximum transformation effect, reaching 85.4%. We have identified 5 actively growing clones of hairy roots with intensive branching, the growth indices of which were 64-75. In the future, they will be transferred to a liquid medium for biomass accumulation and scaling.

Centella asiatica contains various triterpenoids, including madecassoside, asiaticoside, madecassic acid, and asiatic acid. Among them, they are mainly contained in leaf, and among them asiaticoside is known as tissue-specific compound that are mainly accumulated in the leaves. Therefore, in the present study, C. asiatica hairy root cultures were established from petiole and leaf explants using Agrobacterium rhizogenes-mediated transformation. To promote the biosynthesis of these triterpenoids, hairy root cultures were treated with precursors (squalene and pyruvic acid) and elicitors [methyl jasmonate (MeJA) and salicylic acid (SA)] at various concentrations. No significant difference was detected in the growth index of hairy root cultures between precursor feeding and elicitation, except pyruvic acid feeding, suggesting that elicitors do not affect the growth of hairy roots. Treatment with 2.5 mM squalene and 5 mM pyruvic acid enhanced the biosynthesis of target triterpenoids (57.53 and 29.13 mg g−1 DW, respectively) compared with other treatments. However, compared to squalene, MeJA showed a greater effect on the triterpene saponin content in C. asiatica. The highest quantity of triterpenoids (60.25 mg g−1 DW) was produced in hairy root cultures treated with 400 μM MeJA. These results indicate that precursor- and elicitor-treated C. asiatica hairy root cultures rapidly accumulate biomass and could be used to enhance the production of triterpenoids for industrial purposes.

Introduction: Citrullus colocynthis is considered as a precious herb due to its medicinal and nutritional values and also for its ability to produce valuable bioactive compounds such as cucurbitacin E and quercetin. The hairy root systems are valuable tools for scaling-up secondary metabolites and for introducing new beneficial traits into herbs. The present research has aimed to develop a protocol for hairy root culture of C. colocynthis using Agrobacterium rhizogenes. Materials and Methods: After the establishment of the hairy root system, factors such as explant type, bacterial strain, pre-culture period, co-cultivation period, and the use of acetosyringone that affect the efficient transformation of the herb were optimized. Four A. rhizogenes strains (MSU440, A4, A13 and ATCC 15834) and three types of explant (leaf, excised shoot and hypocotyl) were tested. Furthermore, the insertion of transgene into the genome of C. colocynthis was confirmed by polymerase chain reaction analysis. Results: The highest transformation frequency was obtained after the infection of excised shoots by MSU440. Co-cultivation for 48 hours resulted in enhanced transformation frequency, while the results of this research showed that the protocol is better not to include the pre-culturing step. In addition, the presence of acetosyringone in bacterial culture and co-cultivation medium significantly increased the success of C. colocynthis transformation. Conclusions: This study describes an efficient protocol for hairy roots culture of C. colocynthis which can be used for scaling-up the plant active phytochemicals or for genetic manipulations of the plant.

Aim. Determination of the effect of toxic metals on plants is of considerable interest, since contamination of the environment with toxicants is a threat to the safety of agriculture and human health. Vanadium increased content in soil leads to the suppression of plant growth. The aim of the work was to determine the peculiarities of the influence of V(IV) (neutral solution [V4+ cit]) on Artemisia tilesii Ledeb plants in the in vitro model system and to compare this effect on the growth of “hairy” root culture and plants. Methods. Plant shoots and transgenic roots were cultivated on a solidified Murashige and Skoog medium with V(IV) at 1, 5, 10, 50 and 100 mg/L. Mass increase was measured after 4 weeks. Results. It was found that V(IV) at 50 mg/L concentration completely inhibited root formation and led to shoot death. Transgenic roots survived even at 100 mg/L of vanadium. Conclusions. The results of the work demonstrated that A. tilesii “hairy” roots were characterized by greater resistance to V(IV) than control plants. In addition, differences in the sensitivity of different root lines to this compound were detected. This fact probably could be explained by the transformation, the transfer of agrobacterial genes, and changes in the secondary metabolism in transgenic roots. Keywords: vanadium(IV), Artemisia tilesii Ledeb., “hairy” root culture.

Ursolic acid (pentacyclic triterpene acid) and eugenol (phenolic compound), the major secondary metabolites of Ocimum tenuiflorum L., exhibit many pharmacological activities. Due to low quantities of these metabolites in the plant, it was imperative to apply biotechnological approaches for enhancing accumulation of these compounds. Effects of three elicitors namely yeast extract, methyl jasmonate, and salicylic acid were studied on the hairy root cultures induced by infecting leaves of O. tenuiflorum with Agrobacterium rhizogenes strain. The transgenic nature of hairy roots was validated by PCR of rolB gene. The yield of ursolic acid and eugenol responded to the age of the culture, the concentration of the elicitor and the incubation time. The 17-day-old hairy root cultures when treated with yeast extract (50 mg/L) produced the highest amount of ursolic acid of 1.56 mg/g dry weight (5.6-fold higher than control culture) and eugenol content of 0.41 mg/g dry weight (~ 6.0-fold higher than the control) after 8 days of exposure. Methyl jasmonate at 60 mg/L concentration also enhanced the accumulation of ursolic acid up to 1.43 mg/g dry weight (5.0-fold higher than the control) and eugenol up to 0.1 mg/g dry weight (1.55-fold higher than the control) after 8 days of elicitor treatment. Increased elicitor concentration and extended exposure time as well as age of the culture resulted in reduced accumulation of ursolic acid and eugenol in hairy root cultures. Our results demonstrate the importance of age of the culture, different exposure time, and concentration of elicitors in the hairy root culture of O. tenuiflorum for increased production of pharmaceutically important metabolites.

Two lines of Calendula officinalis in vitro hairy root culture, obtained as a result of transformation with the wild type Agrobacterium rhizogenes strain ATCC 15834, were subjected to elicitation by jasmonic acid and chitosan in the context of possible stimulation of biosynthesis, accumulation and secretion of triterpenoids. Jasmonic acid was found to be a very effective elicitor, increasing both the accumulation of oleanolic acid saponins in the hairy root tissue (up to 20-fold) and, in particular, the secretion of these compounds to the medium (up to 113-fold). However, it also promoted inhibition in biosynthesis and accumulation of sterols (approximately by 60%) with some alterations of their profile. Chitosan slightly enlarged the accumulation and secretion of oleanolic acid saponins (up to 3-fold) and simultaneously, increased the sterols total content of (by 5–18%), with some changes of the ratio among individual compounds being observed. The obtained results revealed that the mechanism of action and impact on plant metabolism of the two applied biotic elicitors is different, which have consequences in their efficiency to stimulate triterpenoid production. The trials of stimulation of triterpenoid biosynthesis in plants and plant in vitro cultures often concern the possible competition of pathways leading to sterols and triterpenoids. Some symptoms of the switch at a branch point between primary and secondary triterpenoid metabolism are clearly visible in the response of C. officinalis hairy roots stimulation with jasmonic acid and chitosan.

Panax quinquefolium is a perennial herb of the Araliaceae family native to North America. Its roots have been used in traditional and Chinese medicine. The aim of this study was to determine the phenolic profile of methanolic extracts of P. quinquefolium hairy roots cultivated in flasks and a bioreactor, as well as extracts from the roots of three-year-old field-grown plants. Additionally, the phenol and ginsenoside components of the tested extracts were identified by HPLC, and their antioxidant and cytotoxic properties were evaluated. The antioxidant effect was evaluated by FRAP (ferric reducing antioxidant power), and ABTS ([2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) cation scavenging tests, and their effect on the viability of the glioblastoma cell (T98G) line was measured using the 3-(4,5-dimethylthazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The LC–MS/MS analysis revealed the presence of 16 phenolic compounds identified as phenolic acids (ten compounds) or flavonoids (six compounds). The highest phenol content was observed in the transformed roots of flask-grown P. quinquefolium (1.6 mg g−1 d.w.), followed by these grown in the bioreactor (1.1 mg g−1 d.w.). However, the highest ginsenoside content was found in the roots of the naturally-cultivated plants (67.6 mg g−1 d.w.). The methanolic extracts from hairy root culture of P. quinquefolium appear to have significant antioxidant and cytotoxic potential. Such transformed American ginseng root cultures could represent a potential source of bioactive metabolites for the food or pharmaceutical industry. First time phenolic compound level, antioxidant and cytotoxic properties were estimated in hairy root cultures of Panax quinquefolium cultivated in flasks and nutrient sprinkle bioreactor. Ginsenosides content was also determined.

Feverfew (Tanacetum parthenium) is a medicinal herb that has multiple pharmacologic properties. This plant is rich parthenolide (PN). Hairy roots (HRs) culture is a valuable biotechnological tool for the production of plant secondary metabolites. Furthermore, application of elicitors is one of the most effective strategies to induce the metabolite biosynthesis. This study has investigated the effects of various elicitors on the production of PN in feverfew hairy root culture and the expression of parthenolide synthase (TpPTS) gene. Three elicitors were used separately and in combination, including yeast extract (YE) (2.5 mgL− 1), methyl jasmonate (MJ) (100 µM), Ag+ (100 µM), YE + MJ, Ag+ + MJ, YE + Ag+, and YE + MJ + Ag+ which were added to the 1/2MS medium on day 18 and after 48 h and 96 h, hairy roots were collected to determine the PN content. Total RNA was extracted from hairy roots and used for cDNA synthesis and real-time PCR analysis to investigate the gene expression of TpPTS. The PN content was determined by HPLC. The highest PN content (0.05 mg g− 1 dry weight) was achieved after application of elicitors (in 2.5 mgL− 1 YE and 100 µM MJ) and the highest expression of TpPTS gene also occurred after 96 h in the same treatment. It was concluded that some of the combined elicitors could efficiently increase the parthenolide content of feverfew in vitro culture. The additive effect of elicitors might be due to the separate mechanisms of each substance that is acting in an independent pathway.

Secondary metabolites are a group of chemical compounds in plants which have various functions and applications in different industries such as leather and pharmaceutical products. Artemisia is a genus of important vascular plants which contains many species. There are many chemical compounds in Artemisia which are still unknown. Additionally, hairy root culture in plants is considered to be one of the new techniques applied to production of secondary metabolites which denotes the vast application of this technique. Hairy root culture as a differentiated tissue has been under consideration for many researchers which is because of its genetic stabilization; its rapid growth in a hormone-free medium is also very important. The current study is conducted with the objective of hairy root induction in explant parts of some Artemisia species with various strains of Agrobacterium rhizogenes. The results show that total protein content and enzyme activity during hairy root induction has an ascending procedure. Phenol and flavonoid content have shown considerable increase. The sterol and essential oil have also shown interesting consistency towards each other. To the best of our knowledge, this is the first report of production secondary metabolites and antioxidant activities by hairy root-derived Artemisia. This study may open possibilities for production of the pharmaceutically high-value secondary metabolites.

Transformed hairy root culture in common buckwheat (Fagopyrum esculentum Moench Rubra cultivar) was investigated for accumulation of amino acids and specific flavonoids. Leaves and stems of F. esculentum were used a starting material for induction of hairy roots via the Agrobacterium rhizogenes A4 strain. The transformed lines were confirmed by PCR detection of rol B gene, and their capability to continuously form hairy roots. Three lines from each explant types depending upon growth kinetics were observed. The hairy root lines were used to measure the contents of 17 amino acids and 3 flavonoids. Overall, the hairy root lines exhibited elevated accumulation of semi-essential amino acids such as lysine, isoleucine, valine, histidine and phenylalanine. Content of proline was increased 3–5 times, likely due to the biotic stress reaction induced with A. rhizogenes. Determination of flavonoids by high-performance liquid chromatography, hesperidine and kaempferol-3-rutinoside, were accumulated in hairy root cultures and didn’t detected in non-transformed root. The increase in flavonoids positively correlated with the antioxidant capacity of the hairy root cultures.

Withaferin-A (WA) is a major bioactive compound in the roots of Withania somnifera. It is reported to possess inhibitory effects against breast cancer, liver cancer, melanoma cancer and prostate cancer cells. We developed an elicitation based on the hairy root culture (HRC) protocol for the enhancement of the WA production. Agrobacterium rhizogenes strain R1000 was used for the establishment of hairy root culture. PCR analysis confirmed the integration of the root loci C (rol C) gene in transformed roots. Elicitation of the hairy roots with natural polysaccharides of sodium alginate (SA), k-carrageenan (kC) and chitosan (CH) at various concentrations in the most cases resulted in a higher amount of WA compound. Among the various elicitors, 100 mg L−1 CH resulted 4.03-fold increase in WA production than control. This study is the first report in W. somnifera hairy roots, where the natural polysaccharides are used as elicitors for the large-scale production of WA.

Objective: The present study was aimed to extract betalains from hairy root culture of Beta vulgaris and its use in pharmaceutical formulations as a colorant. Methods: Hairy roots were initiated using different strains of Agrobacterium rhizogenes such as A.2/83, A.20/83, A.4, and LMG 150; LMG 150 was found to initiate a large number of hairy roots, and betalain content was estimated. Paracetamol syrup was prepared using extracted betalains as a colorant at different concentrations of 10 and 30 mg/150 ml. Stability studies were carried out at a different temperatures such as 25°C, 30°C, and 40°C) and light (dark, 1000 and 2000 lux) for 45 days. Results: In case of a concentration of 10 mg/150 ml syrup, the effects of temperature such as 25°C, 30°C, and 40°C and dark condition on the degradation of betalains were found to be 48%, 88%, and 100% in 45 days, respectively. The effects of temperature such as 25°C, 30°C, and 40°C at light 1000 lux on degradations of betalains were found to be 81% and 98% at 25°C and 30°C in 45 days, respectively, and 100% at 40°C in 30 days and at 2000 lux were found to be 100% at 25°C and 30°C in 30 days and 100% at 40°C in 10 days. The similar levels of degradation rate were observed with a concentration of 30 mg/150 ml. Conclusion: Experimental data demonstrated that formulation with betalains exhibited better stability at the dark condition and lower temperature.

For a long time, Cannabis sativa has been used for therapeutic and industrial purposes. Due to its increasing demand in medicine, recreation, and industry, there is a dire need to apply new biotechnological tools to introduce new genotypes with desirable traits and enhanced secondary metabolite production. Micropropagation, conservation, cell suspension culture, hairy root culture, polyploidy manipulation, and Agrobacterium-mediated gene transformation have been studied and used in cannabis. However, some obstacles such as the low rate of transgenic plant regeneration and low efficiency of secondary metabolite production in hairy root culture and cell suspension culture have restricted the application of these approaches in cannabis. In the current review, in vitro culture and genetic engineering methods in cannabis along with other promising techniques such as morphogenic genes, new computational approaches, clustered regularly interspaced short palindromic repeats (CRISPR), CRISPR/Cas9-equipped Agrobacterium-mediated genome editing, and hairy root culture, that can help improve gene transformation and plant regeneration, as well as enhance secondary metabolite production, have been highlighted and discussed.

Rubia yunnanensis is an important medicinal plant with various bioactive secondary metabolites. In order to reduce the dependence on wild populations of the species, we aim to establish in vitro culture system that can produce Rubiaceae-type cyclopeptides (RAs) and quinones. Agrobacterium rhizogenes-mediated transformation of stem segments of in vitro grown R. yunnanensis plants using four A. rhizogenes strains was studied and transformation conditions were optimized. Hairy roots appeared with the highest frequency (68.89%) when stem segments (with leaves) without pre-culture were immersed in A. rhizogenes A4 strain bacterial suspension for 30 min, co-cultured on Murashige and Skoog (MS) solid medium in the dark for three days, and afterwards incubated in darkness. PCR analysis of rolB and rolC genes confirmed transformed nature of six hairy root clones. The hairy roots grew rapidly, especially showing the highest accumulation of biomass in MS liquid medium compared to in vitro grown plants and calli. Histological observation of hairy root revealed anatomical difference in vascular cylinder, where the cells exhibited high mitotic activity characterized by vigorous growth. The UPLC-MS/MS analysis revealed that the amount of RAs in the hairy roots grown in ½MS liquid medium (4.611 μg g−1 DW) was higher than that in in vitro grown plants (0.331 and 4.096 μg g−1 DW for shoots and roots respectively) and calli (1.082 μg g−1 DW), but still far lower than that in the roots of seed-borne plants (80.296 μg g−1 DW). However, the hairy roots accumulated high level of quinones (2320.923 and 5067.801 μg g−1 DW for MS and ½MS liquid media respectively), of the same order of magnitude as the roots of seed-borne plants (7409.973 μg g−1 DW). Hairy root culture of R. yunnanensis, with high accumulation of biomass and production of quinones, may offer an attractive perspective for the production of the RAs and quinones that could be further optimized for pharmaceutical use.

Rindera graeca is a rare endemic plant where in vitro culture has been used in order to investigate bioactive metabolites. Phytochemical study of the in vitro shoots and hairy roots led to the isolation of seven phenolic derivatives and the unusual furano-naphthoquinone rinderol. R. graeca was also analyzed for its pyrrolizidine alkaloids content by LC-MS, and it was found to contain echinatine together with echinatine and rinderine N-oxides. Rinderol, isolated only from in vitro hairy root culture for the first time in the genus, revealed promising bioactivities. It was evaluated in vitro against a panel of microorganisms, showing very strong activity specifically against Gram-positive bacteria (MIC values 0.98 × 10−2–1.18 µg/mL) as well as very interesting antiproliferative effect against the human non-small-cell bronchopulmonary carcinoma cell line NSCLC-N6-L16 and the epidermoid lung cancer cell line A549. These findings were compared with the chemical profile of the plant from nature, while this study is the first to report on the effects of R. graeca extracts obtained from in vitro culture, providing a valuable contribution to the scientific community towards this sustainable method of production of potential bioactive molecules.

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.

The importance of chamomile (Chamomilla recutita)inflorescence is widely known in classical and folk medicine, with the largest group of its effective constituents forming the essential oil (chamazulene, a-bisabolol, α-farnesene, trans-β-farnesene, spathulenol, cis/trans-en-in-dicycloethers). Among cultivated species, the Hungarian BK-2 contains more chamazulene in its essential oil than the German Degumil type, which is mainly cultivated for its a-bisabolol. Both components have important antiinflammatory activities. Wild populations can be easily distinguished from cultivated ones by their high amount of bisaboloides, particularly the flower of Hungarian Szabadkígyós wild type, which contained on average 48 % of the biologically active (-)-a-bisabolol. The population of Szabadkígyós has good salt tolerance which is important owing to global warming, because the proportion of saline areas is increasing worldwide. To keep the genome of Szabadkígyós having high (-)-a-bisabolol content, Szőke and research team used biotechnological methods. Sterile plantlets, were infected byAgrobacterium rhizogenesstrains #A-4, #15834, #R-1601. The hairy root clones possessing the best growing and biosynthetical potential were multiplied for phytochemical investigations. Pharmacologically important compounds of their essential oils were followed in great detail. The amount ofin vitrocultured terpenoids and polyin compounds was compared with that ofin vivoplants. GC-MS studies showed that sterile chamomile cultures generated the most important terpenoid and polyin compounds characteristics of the mother plant. Berkheyaradulene, geranyl-isovalerat and cedrol as new components were identified in these sterile cultures. The main component of hairy root cultures (D/400, D/1, D/100 and Sz/400) was tr-b-farnesene and in addition one new compound: a-selinene was identified. Hairy root culture originated from chamomile collected in Szabadkígyós was intensive increased the essential oil content and pharmacological active compounds: (-) -α-bisabolol and β-eudesmol was also synthetized in large quantity. Furthermore,in vitroorganized cultures were made from this population to obtain propagation material containing numerous active substances.

The secondary metabolites of nightshade and coca plant families contain important compounds known as tropane alkaloids (TAs). TAs possess pharmaceutical properties and are used in the treatment of several neurological disorders such as cardiac arrhythmia, Parkinson’s disease, etc. These compounds are also used as bronchodilators and anesthetics, although their prolonged use has many side effects. Comparing the global consumption rate of the TAs to the production by the original plant source, their yield is too low. Thus, it is important to increase their production. Several TAs such as scopolamine, spermidine, and hyoscyamine have been subjected to the de novo manufacturing processes for their large-scale production. This chapter summarizes the scientific efforts used to scale up the production of TAs for medicinal purposes. Several strategies such as tissue culture, root culture, hairy root culture, and genetically modified microbe (bacteria and yeast)-mediated large-scale production have been described. However, the best strategies are expected to come in near future, which could fulfill the gap of requirement and the production rate of TAs along with their preserved bioactivity.

Medicinal plants are treasures of nature with almost never-ending resource of unlimited, diverse, complex and valuable natural compounds with a variety of pharmacological properties. They are used worldwide by all human beings to maintain and restore good health since ancient times. The cumulative effect of heterogeneous distribution and availability throughout the world, anthropogenic interferences like habitat destruction, overexploitation due to increasing demand, unsustainable harvesting practices from the wild, introduction of exotic species, climatic changes, lack of knowledge about medicinal properties and domestication, less agronomical preference in comparison with food crops, weakening of biodiversity protection laws and/or tendency to undermine these laws by modern socio-economic forces are responsible for the loss of medicinal plant biodiversity. Classical plant tissue culture (PTC)-based biotechnological approaches have been efficiently utilized as sustainable platforms for the conservation of endangered, disease-prone and recalcitrant medicinal plant species via in vitro micropropagation and in vitro production of pharmaceutically important bio-active phytochemicals in different transformed and/or non-transformed cells, organs or regenerated plants that effectively decrease harvesting pressure from wild populations of medicinal plants. Advancement in genomics, transcriptomics, proteomics, nanoscience and synthetic biology in recent times have started to revolutionize medicinal plant research in many dimensions where PTC techniques play very important role for the implementation of these modern fields of science by providing constant aseptic control culture condition and better scope to utilize totipotency in comparison with wild cultivation. Hairy root culture, ploidy engineering, metabolic pathway engineering, transgenic plant development, large-scale production in bioreactors, application of Zinc-finger nucleases, TALENs and CRIPR/Cas9 are promising new biotechnological approaches. In spite of various applications and advantages of the PTC techniques, the broader commercial utilization of in vitro techniques as an important tool for the germplasm conservation of threatened commercially important medicinal plant species via mass proliferation and industrial production of SMs can be challenged by genetic, phenotypic and phytochemical instability often developed by somaclonal variations in long-term in vitro culture. The present paper reviews the recent (from 2010 upto June 2020) achievements in PTC research for conservation of medicinal plants, production of important SMs, and the stability of in vitro cultures along with case studies of two endangered species, Podophyllum hexandrum and Rauvolfia serpentina. There are immense opportunities to produce pharmaceutical important SMs with minimal threat to the biodiversity by the application of suitable conventional and upcoming PTC techniques along with screening for stability.

Rhizobium rhizogenes induces a disease syndrome in infected plants commonly known as hairy roots (HR) production. These unique organs are considered of transgenic nature since they derived after horizontal gene transfer from the bacterial root-inducing (Ri) plasmid to the plant genome. The HR culture represents a major biotechnological tool facilitating the production of plant-derived compounds exhibiting interesting features for pharmaceutical, cosmetics and food industries. Moreover, HR cultures can be used for studying the biosynthetic pathways of plant-derived molecules or investigating interactions occurring during phytoremediation. Among this wide range of HR applications, it is important to point out the possibility to synthesize some antidiabetic compounds which are of special interest because diabetes is one of the major and frequently occurring debilitating diseases of the human body in the world. The use of available synthetic antidiabetic drugs has several limitations, including drug resistance, side effects, or toxicity. Therefore, the availability of new antidiabetic drugs from a natural source is of big importance. Stevia rebaudiana Bertoni is one of the promising plant species, constituting the source of desired plant molecules. This plant accumulates steviol glycosides (SGs), which are responsible for the sweet flavour of stevia leaves. These secondary metabolites are considered metabolically inert and therefore could be a safe substitute for sugar for diabetic people. In this chapter, we would like to present recent achievements in the range of antidiabetic substances production via genetic transformation of plants using R. rhizogenes strains. In this context, a key role of the so-called rol genes, carried by Ri plasmids, in the biosynthesis of various secondary metabolites is also discussed. Our aim is to bring closer the issue of HR cultures and their big potential in the pharmacognosy of antidiabetic plants.

Ipomoea aquatica Forsk. (Convolvulaceae) is a commonly grown vegetable in the Americas, Africa and especially Southeast Asia, including India. Due to the presence of numerous secondary metabolites, this plant has considerable therapeutic as well nutraceutical value and is categorized among highly prioritized but neglected leafy vegetables. Proper identification of the higher quality genotypes of I. aquatica will help scientists explore major genes to develop future high-quality varieties. Therefore, an integrated approach combining traditional and molecular plant breeding should be carried out to strengthen future breeding programs. Identification of traits controlling genes by extensive database searching with bioinformatics, followed by genomics and transgenic approaches, opens a new possibility to use these beneficial vegetables as potent nutraceuticals, especially in developing countries where malnutrition is a matter of concern. Application of plant cell culture technique can be an attractive field of research for this plant species. In this context micropropagation is the best choice for producing year around pilot-scale production within a short time span. In vitro plantlets can also be conserved as artificial seed to maintain elite plant lines with augmented secondary metabolites. Screening by the use of hairy root culture under photoautotrophic condition to detect contaminants and pollution can assure cultivars are safe to consume. This chapter presents an overview of the origin, distribution, botanical classification, breeding through classical and molecular approaches, tissue-culture practices like rapid micropropagation for high frequency regeneration, use of elite clones and conservation by alginate entrapment, prospects of using hairy root culture, recent developments and future scope of biotechnology and molecular biology using bioinformatics and transgenic approaches and their application for improvement of I. aquatica.

Hairy root (HR) culture, a successful biotechnology combining in vitro tissue culture with recombinant DNA machinery, is intended for the genetic improvement of plants. This technology has been put to use since the last three decades for genetic advancement of medicinal and aromatic plants and also to harvest the economical products in the form of secondary metabolites that are significantly important for their ethnobotanical and pharmacological properties. It also provides an efficient way out for the quicker extraction and quantification of the valuable phytochemicals. The current review provides an account of the in vitro HR culture technology and its wide-scale applications in the field of research as well as in pharmaceutical industries. Different facets of HR with respect to the culture establishment, phytochemical production as well as research investigations concerning the areas of gene manipulation, biotransformation of the secondary metabolites, phytoremediation, their industrial utilisations and different problems encountered during the application of this technology have been covered in this appraisal. Eventually, an idea has been provided on HR about the recent trends on the progress of this technology that may open up newer prospects in near future and calls for further research and explorations in this field. KEY POINTS: • Genetic engineering-based HR culture aims towards enhanced secondary metabolite production. • This review explores an insight in the HR technology and its multi-faceted approaches. • Up-to-date ground-breaking research applications and constraints of HR culture are discussed.

Echium plantagineum L. (Boraginaceae) is an invasive species in Australia and contains medicinal shikonins in its roots. In this study, the hairy root lines of E. plantagineum were established using Agrobacterium rhizogenes strain ATCC15834 and confirmed by the amplification of the rolB gene. Results showed significant difference in shikonin production between the hairy root lines in the 1/2B5 and M9 media. The biomass of the lines in the 1/2B5 medium was fivefold of that in the M9 medium. However, the components of detected shikonins were similar in these two liquid media. By contrast, different accumulation profiles appeared in the hairy root lines. HPLC analysis revealed the presence of nine possible related compounds, including shikonins, and acetylshikonin was the most abundant shikonin derivative. The content of acetylshikonin in the 1/2B5 medium (36.25 mg/L on average) was twofold of that in the M9 medium. Our results showed that the hairy root cultures of E. plantagineum can be used in enhancing the production of potential pharmaceutical compounds, such as acetylshikonin.

White mulberry (Morus alba L.) root was reported to contain potential tyrosinase inhibitors such as norartocarpetin, streppogenin, oxyresveratrol (5, 7, 2′, 4′-tetrahydroxy-flavavone) and 2-arylbenzofuran. This study aims at investigating Morus alba L. hairy root culture to create a source of tyrosinase inhibitors for skin-whitening application. Morus alba L. leaf (including mesophyll and petiole) and stem explants were randomly wounded and inoculated with Agrobacterium rhizogenes ATCC 15834 in 10-40 minutes, and coculturing for 4 days. After inoculation, Agrobacterium rhizogenes were removed by using MS medium adding 200mg/L cefotaxime as antibiotics. Then, fresh inducted hairy roots were grown in nonphytohormone liquid medium (MS, White, Gamborg B5 or WPM) on a shaker (80rpm) at 25 ±1°C. Inducted hairy roots were tested for the present of rolB by PCR analysis and its effect on tyrosinase activity using a 96-well plate reader. The suitable explants for hairy root induction are leaves, especially petioles in 10 minutes (root induction frequency: 83%). After induction, hairy roots on MS was grown more efficiently (increased 3.933 times in FW) than on WPM, B5 and White medium. Inducted hairy roots were successfully transgenes by the present of rolB in their genome and had the same anti-tyrosinase activity as the natural roots without significance differences (IC50: 3.860 and 3.597 μg/mL, respectively). Cultured Morus alba L. hairy roots are potential source of tyrosinase inhibitors for future skin-whitening ingredient.

The in vitro tissue culture of medicinal plants is considered as a potential source for plant-derived bioactive secondary metabolites. The in vitro tissue culture of American ginseng has wide commercial applications in pharmaceutical, nutraceutical, food, and cosmetic fields with regard to the production of bioactive compounds such as ginsenosides and polysaccharides. This review highlights the recent progress made on different types of tissue culture practices with American ginseng, including callus culture, somatic embryo culture, cell suspension culture, hairy root culture, and adventitious root culture. The tissue culture conditions for inducing ginseng callus, somatic embryos, cell suspension, hairy roots, and adventitious roots were analyzed. In addition, the optimized conditions for increasing the production of ginsenosides and polysaccharides were discussed. This review provides references for the use of modern biotechnology to improve the production of bioactive compounds from American ginseng, as well as references for the development and sustainable utilization of American ginseng resources.

The medicinal plant Catharanthus roseus produces numerous secondary metabolites of interest for the treatment of many diseases – most notably for the terpene indole alkaloid (TIA) vinblastine, which is used in the treatment of leukemia and Hodgkin's lymphoma. Historically, methyl jasmonate (MeJA) has been used to induce TIA production, but in the past, this has only been investigated in whole seedlings, cell culture, or hairy root culture. This study examines the effects of the phytohormones MeJA and ethylene on the induction of TIA biosynthesis and accumulation in the shoots and roots of 8‐day‐old seedlings of two varieties of C. roseus. Using LCMS and RT‐qPCR, we demonstrate the importance of variety selection, as we observe markedly different induction patterns of important TIA precursor compounds. Additionally, both phytohormone choice and concentration have significant effects on TIA biosynthesis. Finally, our study suggests that several early‐induction pathway steps as well as pathway‐specific genes are likely to be transcriptionally regulated. Our findings highlight the need for a complete set of'omics resources in commonly used C. roseus varieties and the need for caution when extrapolating results from one cultivar to another.