Hairy Root Lines Research Articles

Echium acanthocarpum hairy root cultures, a suitable system for polyunsaturated fatty acid studies and production

BackgroundThe therapeutic and health promoting role of highly unsaturated fatty acids (HUFAs) from fish, i.e. eicosapentaenoic acid (EPA, 20:5n-3) and docosahexaenoic acid (DHA, 22:6n-3) are well known. These same benefits may however be shared by some of their precursors, the polyunsaturated fatty acids (PUFAs), such as stearidonic acid (SDA, 18:4 n-3). In order to obtain alternative sources for the large-scale production of PUFAs, new searches are being conducted focusing on higher plants oils which can contain these n-3 and n-6 C18 precursors, i.e. SDA and GLA (18:3n-6, γ-linolenic acid).ResultsThe establishment of the novel Echium acanthocarpum hairy root cultures represents a powerful tool in order to research the accumulation and metabolism of fatty acids (FAs) in a plant particularly rich in GLA and SDA. Furthermore, this study constitutes the first example of a Boraginaceae species hairy root induction and establishment for FA studies and production. The dominant PUFAs, 18:2n-6 (LA, linoleic acid) and 18:3n-6 (GLA), accounted for about 50% of total FAs obtained, while the n-3 PUFAs, 18:3n-3 (ALA, α-linolenic acid) and 18:4n-3 (SDA), represented approximately 5% of the total. Production of FAs did not parallel hairy root growth, and the optimal productivity was always associated with the highest biomass density during the culture period. Assuming a compromise between FA production and hairy root biomass, it was determined that sampling times 4 and 5 gave the most useful FA yields. Total lipid amounts were in general comparable between the different hairy root lines (29.75 and 60.95 mg/g DW), with the major lipid classes being triacylglycerols. The FAs were chiefly stored in the hairy roots with very minute amounts being released into the liquid nutrient medium.ConclusionsThe novel results presented here show the utility and high potential of E. acanthocarpum hairy roots. They are capable of biosynthesizing and accumulating a large range of polyunsaturated FAs, including the target GLA and SDA fatty acids in appreciable quantities.

Characterization of glycosyltransferases responsible for salidroside biosynthesis in Rhodiola sachalinensis

Salidroside, the 8- O-β- d-glucoside of tyrosol, is a novel adaptogenic drug extracted from the medicinal plant Rhodiola sachalinensis A. Bor. Due to the scarcity of R. sachalinensis and its low yield of salidroside, there is great interest in enhancing production of salidroside by biotechnological manipulations. In this study, two putative UDP-glycosyltransferase (UGT) cDNAs, UGT72B14 and UGT74R1, were isolated from roots and cultured cells of methyl jasmonate (MeJA)-treated R. sachalinensis, respectively. The level of sequence identity between their deduced amino acid sequences was ca. 20%. RNA gel–blot analysis established that UGT72B14 transcripts were more abundant in roots, and UGT74R1 was highly expressed in the calli, but not in roots. Functional analysis indicated that recombinant UGT72B14 had the highest level of activity for salidroside production, and that the catalytic efficiency ( V max/ K m) of UGT72B14 was 620% higher than that of UGT74R1. The salidroside contents of the UGT72B14 and UGT74R1 transgenic hairy root lines of R. sachalinensis were also ∼420% and ∼50% higher than the controls, respectively. UGT72B14 transcripts were mainly detected in roots, and UGT72B14 had the highest level of activity for salidroside production in vitro and in vivo.

Metabolic engineering tanshinone biosynthetic pathway in Salvia miltiorrhiza hairy root cultures

Tanshinone is a group of active diterpenes widely used in treatment of cardiovascular diseases. Here, we report the introduction of genes encoding 3-hydroxy-3-methylglutaryl CoA reductase (HMGR), 1-deoxy- d-xylulose-5-phosphate synthase (DXS) and geranylgeranyl diphosphate synthase (GGPPS) involved in tanshinone biosynthesis into Salvia miltiorrhiza hairy roots by Agrobacterium-mediated gene transfer technology. Overexpression of SmGGPPS and/or SmHMGR as well as SmDXS in transgenic hairy root lines can significantly enhance the production of tanshinone to levels higher than that of the control ( P<0.05). SmDXS showed much more powerful pushing effect than SmHMGR in tanshinone production, while SmGGPPS plays a more important role in stimulating tanshinone accumulation than the upstream enzyme SmHMGR or SmDXS in S. miltiorrhiza. Co-expression of SmHMGR and SmGGPPS resulted in highest production of tanshinone (about 2.727 mg/g dw) in line HG9, which was about 4.74-fold higher than that of the control (0.475 mg/g dw). All the tested transgenic hairy root lines showed higher antioxidant activity than the control. To our knowledge, this is the first report on enhancement of tanshinone content and antioxidant activity achieved through metabolic engineering of hairy roots by push–pull strategy in S. miltiorrhiza.

Overexpression of an apoplastic peroxidase gene CrPrx in transgenic hairy root lines of Catharanthus roseus

Peroxidases are a family of isoenzymes found in all higher plants and are known to be involved in a broad range of physiological processes. However, very little information is available concerning their role in Catharanthus roseus. The present study describes the impact of both overexpression and suppression of a peroxidase gene, CrPrx in C. roseus transgenic hairy root lines. Real-time PCR analysis in 35S-CrPrx and CrPrx-RNAi transgenic lines indicated differential transcript profile for peroxidases as well as for genes and regulators involved in MIA (monoterpenoid indole alkaloid) pathway of C. roseus. Comparative analysis revealed that MIA pathway genes showing elevated levels of expression in 35S-CrPrx transgenic lines showed a significant reduction in their transcript level in CrPrx-RNAi transgenic lines. Metabolite analysis detected higher levels of ajmalicine and serpentine accumulation in overexpressed lines. It was observed that all overexpressed transgenic lines produced more amount of H(2)O(2). These results indicate a role of CrPrx gene in the regulation of MIA pathway genes and regulators, thus affecting the production of specific alkaloids.

Sonication-assisted Agrobacterium rhizogenes-mediated transformation of Verbascum xanthophoeniceum Griseb. for bioactive metabolite accumulation

An efficient protocol for the establishment of transformed root culture of Verbascum xanthophoeniceum using sonication-assisted Agrobacterium rhizogenes-mediated transformation is reported. Only 10days after the inoculation with A. rhizogenes ATCC 15834 and 45s ultrasound exposure, hairy roots appeared on 75% of the Verbascum leaves. Ten hairy root lines were isolated, although only half of them were free of bacterial contamination and started growing when excised from mother explants. The transgenic nature of the most vigorously growing hairy root clones (VX1 and VX6) was confirmed by polymerase chain reaction. Under submerged cultivation both hairy root clones accumulated high biomass amounts (12.8 and 14.3gL(-1), respectively) and significant amounts of bioactive phenylethanoid glycoside verbascoside (over 6-times more than in mother plant leaves). LC-APCI-MS analyses confirmed verbascoside accumulation in hairy root clones along with three other phenylethanoid glycosides (forsythoside B, leucosceptoside B and martynoside) and an iridoid glycoside aucubin. This is the first report on the induction of hairy roots of Verbascum plants.

Development of a transgenic hairy root system in jute (Corchorus capsularis L.) with gusA reporter gene through Agrobacterium rhizogenes mediated co-transformation

Transgenic hairy root system is important in several recalcitrant plants, where Agrobacterium tumefaciens-mediated plant transformation and generation of transgenic plants are problematic. Jute (Corchorus spp.), the major fibre crop in Indian subcontinent, is one of those recalcitrant plants where in vitro tissue culture has provided a little success, and hence, Agrobacterium-mediated genetic transformation remains to be a challenging proposition in this crop. In the present work, a system of transgenic hairy roots in Corchorus capsularis L. has been developed through genetic transformation by Agrobacterium rhizogenes harbouring two plasmids, i.e. the natural Ri plasmid and a recombinant binary vector derived from the disarmed Ti plasmid of A. tumefaciens. Our findings indicate that the system is relatively easy to establish and reproducible. Molecular analysis of the independent lines of transgenic hairy roots revealed the transfer of relevant transgenes from both the T-DNA parts into the plant genome, indicating the co-transformation nature of the event. High level expression and activity of the gusA reporter gene advocate that the transgenic hairy root system, thus developed, could be applicable as gene expression system in general and for root functional genomics in particular. Furthermore, these transgenic hairy roots can be used in future as explants for plantlet regeneration to obtain stable transgenic jute plants.

The expression of 1-deoxy- d-xylulose synthase and geraniol-10-hydroxylase or anthranilate synthase increases terpenoid indole alkaloid accumulation in Catharanthus roseus hairy roots

The terpenoid indole alkaloid (TIA) pathway in Catharanthus roseus produces two important anticancer drugs, vinblastine and vincristine, in very low yields. This study focuses on overexpressing several key genes in the upper part of the TIA pathway in order to increase flux toward downstream metabolites within hairy root cultures. Specifically, we constructed hairy root lines with inducible overexpression of 1-deoxy- D-xylulose synthase (DXS) or geraniol-10-hydroxylase (G10H). We also constructed hairy root lines with inducible expression of DXS and anthranilate synthase α subunit (ASA) or DXS and G10H. DXS overexpression resulted in a significant increase in ajmalicine by 67%, serpentine by 26% and lochnericine by 49% and a significant decrease in tabersonine by 66% and hörhammericine by 54%. Co-overexpression of DXS and G10H caused a significant increase in ajmalicine by 16%, lochnericine by 31% and tabersonine by 13%. Likewise, DXS and ASA overexpression displayed a significant increase in hörhammericine by 30%, lochnericine by 27% and tabersonine by 34%. These results point to the need for overexpressing multiple genes within the pathway to increase the flux toward vinblastine and vincristine.

Response of Soybean Pathogens to Glyceollin

Plants recognize invading pathogens and respond biochemically to prevent invasion or inhibit colonization in plant cells. Enhancing this response in crop plants could improve sustainable methods to manage plant diseases. To enhance disease resistance in soybean, the soybean phytoalexin glyceollin was assessed in soybean hairy roots of two soybean genotypes, Spencer and PI 567374, transformed with either soybean isoflavone synthase (IFS2) or chalcone synthase (CHS6) genes that were inoculated with the soybean pathogens Diaporthe phaseolorum var. meridionales, Macrophomina phaseolina, Sclerotinia sclerotiorum, and Phytophthora sojae. The hairy-root-transformed lines had several-fold decreased levels of isoflavone daidzein, the precursor of glyceollin, and considerably lower concentrations of glyceollin induced by pathogens measured 5 days after fungal inoculation compared with the nontransformed controls without phenolic transgenes. M. phaseolina, P. sojae, and S. sclerotiorum grew much more on IFS2- and CHS6-transformed roots than on control roots, although there was no significant difference in growth of D. phaseolorum var. meridionales on the transformed hairy-root lines. In addition, glyceollin concentration was lower in D. phaseolorum var. meridionales-inoculated transformed and control roots than roots inoculated with the other pathogens. Glyceollin inhibited the growth of D. phaseolorum var. meridionales, M. phaseolina, P. sojae, S. sclerotiorum, and three additional soybean pathogens: Cercospora sojina, Phialophora gregata, and Rhizoctonia solani. The most common product of glyceollin conversion or degradation by the pathogens, with the exception of P. sojae, which had no glyceollin degradation products found in the culture medium, was 7-hydroxyglyceollin.

Effect of loss of T-DNA genes on MIA biosynthetic pathway gene regulation and alkaloid accumulation in Catharanthus roseus hairy roots

Hairy roots are generated by integration of T-DNA in host plant genome from root inducing (Ri) plasmid of Agrobacterium rhizogenes and have been utilized for production of secondary metabolites in different plant systems. In Catharanthus roseus, hairy roots are known to show different morphologies, growth patterns, and alkaloid contents. It is also known that during transformation, there is a differential loss of a few T-DNA genes. To decipher the effect of loss of T-DNA genes on the various aspects of hairy roots, ten hairy root clones were analyzed for the presence or absence of T-DNA genes and its implications. It was found that the loss of a few ORFs drastically affects the growth and morphological patterns of hairy roots. The absence of T(R)-DNA from hairy roots revealed increased transcript accumulation and higher alkaloid concentrations, whereas callusing among hairy root lines led to decreased transcript and alkaloid accumulation. Significantly higher expression of MIA biosynthetic pathway genes and low abundance of regulator transcripts in hairy root clones in comparison with non-transformed control roots were also observed. This study indicates that it is not only the integration of T-DNA at certain region of host plant genome but also the presence or absence of important ORFs that affects the expression patterns of MIA biosynthetic pathway genes, regulators, and accumulation of specific alkaloids.

Embryogenic responses of Beta vulgaris L. callus induced from transgenic hairy roots

Agrobacterium rhizogenes A4M70GUS-mediated transformation of two local breeding lines of sugar beet was obtained using 4-week-old seedlings. Root formation efficiency was 61.54% for SBa genotype and 36.36% for SBb genotype. Five highly proliferated hairy root lines have been established in liquid hormone-free MS medium. Transgenic nature of the hairy root clones was evaluated by GUS assay, PCR and RT-PCR analyses. Hairy root-derived calli were induced using different plant growth regulators (PGRs): auxin, auxin/cytokinin and cytokinin. The best callus induction response was achieved on MS medium containing both 1 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D) and 1 mg/l thidiazuron (TDZ). Globular embryo-like structures were observed in friable callus after its prolonged cultivation on MS medium supplemented with TDZ and giberellic acid (GA3) at 1 mg/l each, followed by growth on MS medium containing 1% glucose and 0.5 mg/l 2,3,5-triiodobenzoic acid (TIBA). Histological analysis revealed somatic embryos at different stages of development in hairy root-derived callus of sugar beet.

Upregulation of phytosterol and triterpene biosynthesis in Centella asiatica hairy roots overexpressed ginseng farnesyl diphosphate synthase

Farnesyl diphosphate synthase (FPS) plays an essential role in organ development in plants. However, FPS has not previously been identified as a key regulatory enzyme in triterpene biosynthesis. To elucidate the functions of FPS in triterpene biosynthesis, C. asiatica was transformed with a construct harboring Panax ginseng FPS (PgFPS)-encoding cDNA coupled to the cauliflower mosaic virus 35S promoter. Higher levels of CaDDS (C. asiatica dammarenediol synthase) and CaCYS (C. asiatica cycloartenol synthase) mRNA were detected in all hairy root lines overexpressing when compared with the controls. However, no differences were detected in any expression of the CaSQS (C. asiatica squalene synthase) gene. In particular, the upregulation of CaDDS transcripts suggests that FPS may result in alterations in triterpene biosynthesis capacity. Squalene contents in the T17, T24, and T27 lines were increased to 1.1-, 1.3- and 1.5-fold those in the controls, respectively. The total sterol contents in the T24 line were approximately three times higher than those of the controls. Therefore, these results indicated that FPS performs a regulatory function in phytosterol biosynthesis. To evaluate the contribution of FPS to triterpene biosynthesis, we applied methyl jasmonate as an elicitor of hairy roots expressing PgFPS. The results of HPLC analysis revealed that the content of madecassoside and asiaticoside in the T24 line was transiently increased by 1.15-fold after 14 days of MJ treatment. This result may indicate that FPS performs a role not only in phytosterol regulation, but also in triterpene biosynthesis.

Induced biosynthesis of resveratrol and the prenylated stilbenoids arachidin-1 and arachidin-3 in hairy root cultures of peanut: Effects of culture medium and growth stage

Previously, we have shown that hairy root cultures of peanut provide a controlled, sustainable and scalable production system that can be induced to produce stilbenoids. However to leverage peanut hairy roots to study the biosynthesis of this polyphenolic biosynthetic pathway, growing conditions and elicitation kinetics of these tissue cultures must be defined and understood. To this end, a new peanut cv. Hull hairy root (line 3) that produces resveratrol and its prenylated analogues arachidin-1 and arachidin-3 upon sodium acetate-mediated elicitation was established. Two culture media were compared for impact on root growth and stilbenoid biosynthesis/secretion. The levels of ammonium, nitrate, phosphate and residual sugars were monitored along growth and elicitation period. A modified MS (MSV) medium resulted in higher root biomass when compared to B5 medium. The stilbenoid profile after elicitation varied depending on the age of the culture (6, 9, 12, and 15-day old). After elicitation at day 9 (exponential growth in MSV medium), over 90% of the total resveratrol, arachidin-1 and arachidin-3 accumulated in the medium. Our studies demonstrate the benefits of the hairy root culture system to study the biosynthesis of stilbenoids including valuable prenylated polyphenolic compounds.

Establishment of hairy root lines and analysis of gentiopicroside in the medicinal plant Gentiana macrophylla

The leaf explants from the eight-week-old Gentiana macrophylla Pall sterile seedlings were precultured for three days on Murashige and Skoog (MS) agar medium with 1 mg/l of 6-benzyladenine, and then incubated in MS liquid medium containing Agrobacterium rhizogenes R1000 for 2 h with shaking. After 10 days of culture on hormone-free MS agar medium with 500 mg/l cefotaxime under illumination of 300 μmol/(m2 s) with a 16-h photoperiod at 24°C, up to 18.3% of the infected explants produced typical hairy roots (HRs). Finally, three stable HR lines with no morphologically visible differences were obtained. The HR lines could vigorously grow and propagate on/in hormone-free half-strength MS solid/liquid medium. Moreover, they changed from white to green when moved from darkness to the light and spontaneously produced calli, which were able later to produce adventitious buds. PCR analysis revealed that three HR lines, which were undergone to continuous subculturing over two years, still contained the rolC gene from the A. rhizogenes Ri plasmid. HPLC revealed the HR line possessing a certain capacity of synthesizing gentiopicroside (0.11 mg/g dry wt).

Growth kinetics and ginsenosides production in transformed hairy roots of American ginseng—Panax quinquefolium L

A thin, profusely branched, fast growing hairy root line of Panax quinquefolium (American ginseng) was established by co-culturing epicotyl explants with a wild type strain of Agrobacterium rhizogenes. The transformed roots grew by over 10-fold from the initial inoculum within 8 weeks. The crude ginsenosides content in the roots was about 0.2 g/g dry wt level up to the 10th week of culture. Ginsenosides Rb2, Rd, Re, Rf and Rg1 constituted 47-49% of the crude saponin fraction between 6 and 8 weeks of growth whereas, Rc ginsenoside was accumulated only after 9th weeks when the biomass started receding. PCR amplification analysis of the hairy roots confirmed their transgenic nature by showing the presence of Ri-TL DNA with rolA, rolB and rolC genes in their genome.

Efficient Production of Gossypol from Hairy Root Cultures of Cotton (Gossypium hirsutum L.)

A protocol for induction and establishment of Agrobacterium rhizogenes mediated hairy root culture of Gossypium hirsutum was developed through infection with the A4 strain and co-cultivation on hormone-free semi-solid MS medium with B5 vitamins. It resulted in the emergence of hairy roots from the leaf explants, 21 days after infection. The transformation of hairy roots was established by PCR amplification of rol B and rol C genes of the Ri plasmid. All root lines expressed gossypol, although distinct inter-clonal quantitative variations were noticed. Five independent hairy root lines were studied for their growth kinetics as well as gossypol production. The yield potentials of one of them superseded others, as well as the non-transformed, in-vitro grown control roots. The content of gossypol in hairy roots reached a level of 2.43 mg/g DW. It was 4.5 times higher than in vitro and 1.47 times higher than in vivo grown roots of G. hirsutum. Selection of high gossypol producing hairy root lines of G. hirsutum can provide an alternative source for large-scale production of gossypol.

Molecular cloning and characterization of two tropinone reductases in Anisodus acutangulus and enhancement of tropane alkaloid production in AaTRI‐transformed hairy roots

Tropane alkaloids are used medicinally as anticholinergic agents with increasing market demand, so the improvement and production of active components from medicinal plants using molecular biotechnology show great potential for applications that should benefit human healthcare. Two tropinone reductases constitute a branching point in the biosynthesis of tropane alkaloids. In the present paper, we report for the first time the cloning and characterization of two fulllength cDNAs encoding TRI (tropinone reductase I) (GenBank accession number EU424321) and TRII (tropinone reductase II) (GenBank(R) accession number EU424322) from the solanaceous plant Anisodus acutangulus by rapid amplification of cDNA ends. Sequence comparison indicated that AaTRI (A. acutangulus TRI) and AaTRII (A. acutangulus TRII) had high homology with other tropinone reductases from Hyoscyamus niger, Datura stramonium etc., but AaTRI and AaTRII showed identity of only 60.8%. Phylogenetic-tree analysis showed that AaTRI and AaTRII belong to different clusters and have the closest relationship with H. niger TRI and TRII respectively. Expression-pattern analysis showed that AaTRI and AaTRII were expressed in all tissues tested, including root, stem and leaf, but the transcript level of AaTRI was much lower than AaTRII. Expression of AaTRI and AaTRII could be enhanced by methyl jasmonate, with a weak effect for AaTRI and a strong effect for AaTRII. AaTRI-transformed hairy-root lines were accompanied by a mean 1.87-fold higher level of hyoscyamine and a mean 8-fold higher level of scopolamine compared with control roots, indicating that AaTRI is a promising target for genetic engineering to increase tropane alkaloid in A. acutangulus.

Mass production and application of activation tagged hairy root lines for functional genomic of secondary metabolism in ginseng

Activation tagging that uses T-DNA vectors containing multimerized transcriptional enhancers from the cauliflower mosaic virus (CaMV) 35S gene is a powerful tool to determine gene function in plants. This approach has been successfully applied in screening various types of mutations and cloning the corresponding genes. We generated an activation tagged hairy root pool of ginseng (Panax ginseng C.A. Meyer) in an attempt to isolate genes involved in the biosynthetic pathway of ginsenoside (triterpene saponin), which is known as the major active ingredient of the root. Quantitative and qualitative variation of ginsenoside in activation tagged hairy root lines were profiled using LC/MS. Metabolic profiling data enabled selection of a specific hairy root line which accumulated ginsenoside at a higher level than other lines. The relative expression level of several genes of triterpene biosynthetic pathway in the selected hairy root line was determined by real time RT-PCR. Overall results suggest that the activation tagged ginseng hairy root system described in this study would be useful in isolating genes involved in a complex metabolic pathway from genetically intractable plant species by metabolic profiling.

Metabolic engineering of indole glucosinolates in Chinese cabbage hairy roots expressing Arabidopsis CYP79B2, CYP79B3, and CYP83B1

Indole glucosinolates (IG), a group of secondary metabolites found almost extensively in the order Brassicales, play an important role in the interaction between plant and insect or microorganism. In order to explore the possibility of IG metabolic engineering in Chinese cabbage hairy roots, three Arabidopsis cDNAs CYP79B2, CYP79B3, and CYP83B1 combined with rolABC were introduced into Chinese cabbage by Agrobacterium-mediated transformation. Overexpression of CYP79B2, CYP79B3, or CYP83B1 alone did not affect the accumulation levels of IG in transgenic hairy roots. However, when CYP83B1 was overexpressed together with CYP79B2 and/or CYP79B3, some of the transgenic hairy roots accumulated higher levels of glucobrassicin (GBC) or 4-methoxy glucobrassicin (4-OMeGBC) than control hairy root line carrying rolABC vector. With regard to the IG accumulation, overexpression of all three cDNAs showed no better than overexpression of both cDNAs. Both 4-OMeGBC and neoglucobrassicin (neo-GBC) were found to be the main components of IG that comprise about 90% of total IG in all types of Chinese cabbage hairy root lines. In transgenic hairy root lines rB3B1-8 and rB2B1B3-5, 4-OMeGBC increased to 2 and 1.5-fold, while neo-GBC decreased to 0.5 and 0.6-fold, respectively. This suggests that an increased production of 4-OMeGBC causes a reduction of neo-GBC level since the two types derive from a common precursor GBC. However, in terms of the total IG level, the transgenic hairy roots did not show significant differences from controls.

Rhinacanthin production by four hairy root lines of Rhinacanthus nasutus (L.) Kurz

Rhinacanthus nasutus (L.) Kurz is a medicinal plant used in Southeast Asia for treatment of several skin diseases [1]. The plant extract possessed several pharmacological activities such as antifungal and antiviral activities [2]. The naphthoquinone compounds, namely rhinacanthins, are major chemical constituents in this plant. In this study, technique of hairy roots induction by Agrobacterium rhizogenes was used for increasing the rhinacanthin level. Four hairy root lines of R. nasutus, including HR11325, HR13332, HR13333 and HR15834, were induced on the leaf explants by A. rhizogenes strains ATCC 11325, 13332, 13333 and 15834, respectively. Transformation percentages were 55%, 25%, 70% and 60%, respectively. The fragments of rolB and rolC genes were observed in all hairy root lines using PCR technique indicating the successful integration of the T-DNA fragment of Ri plasmid of A. rhizogenes to the genome of the hairy roots. Rhinacanthin production from these hairy roots was determined using HPLC technique. HR11325 produced rhinacanthin-C as a major compound together with rhinacanthin-D, and -N with the yields of 2.163, 0.042 and 0.006% w/w, respectively. In contrast, HR13332, HR13333 and HR15384 contained only rhinacanthin-C and -D. The amount of rhinacanthin-C in these three hairy roots were 0.843, 0.824, and 1.148% w/w, while those of rhinacanthin-D were 0.039, 0.012 and 0.017% w/w, respectively.

Hairy Root Cultures of Taxus cuspidatafor Enhanced Production of Paclitaxel

Hairy root cultures were established by transforming seedlings of Taxus cuspidate Sieb. Et Zucc (Korean yew) using Agrobacterium rhizogenes. Initially, 107 lines of hairy roots were induced by infection with 3 strains of Agrobacterium (R1000, A4, and 15384), however, only three lines generated from the R1000 strain were stably grown on hormone-free media over 10 months of successive cultures. These lines were designated as RC11104, RC11105 and RC11106. The hairy root line RC 11106 was selected for further experiments for taxol production based on its growth properties. Upon methyl jasmonate treatment, the RC11106 Une accumulated 52.5 mg/L of taxol over 2 weeks of incubation at a 20-L culture scale.