Study of artemisinin and sugar accumulation in Artemisia vulgaris and Artemisia dracunculus “hairy” root cultures

We studied the effect of genetic transformation on biologically active compound (artemisinin and its co-products (ART) as well as sugars) accumulation in Artemisia vulgaris and Artemisia dracunculus “hairy” root cultures. Glucose, fructose, sucrose, and mannitol were accumulated in A. vulgaris and A. dracunculus “hairy” root lines. Genetic transformation has led in some cases to the sugar content increasing or appearing of nonrelevant for the control plant carbohydrates. Sucrose content was 1.6 times higher in A. vulgaris “hairy” root lines. Fructose content was found to be 3.4 times higher in A. dracunculus “hairy” root cultures than in the control roots. The accumulation of mannitol was a special feature of the leaves of A. vulgaris and A. dracunculus control roots. A. vulgaris “hairy” root lines differed also in ART accumulation level. The increase of ART content up to 1.02?mg/g DW in comparison with the nontransformed roots (up to 0.687?mg/g DW) was observed. Thus, Agrobacterium rhizogenes-mediated genetic transformation can be used for obtaining of A. vulgaris and A. dracunculus “hairy” root culture produced ART and sugars in a higher amount than mother plants.     

Flavonoid content and antioxidant activity of Artemisia vulgaris L. “hairy” roots

We investigated the effect of Agrobacterium rhizogenes-mediated transformation on antioxidant activity of Artemisia vulgaris “hairy” roots. It appeared that transformation may increase flavonoid content as well as DPPH-scavenging activity and ability to reduce Fe³⁺ as compared to the non-transformed plants. Some “hairy” roots accumulated flavonoids up to 73.1?±?10.6?mg RE/g DW (while the amount of flavonoids in the leaves of non-transformed plants was up to 49.4?±?5.0?mg RE/g DW). DPPH-scavenging activity of some “hairy” root lines was 3–3.8 times higher than such one of the roots of the control plants. The Fe³⁺-reducing power of most transgenic root extracts exceeded such power of the extracts of the roots of the control plants. The decrease in SOD activity was found in the most “hairy” root lines compared to the control roots. The increase of flavonoid content correlated with the increase of ability of extracts to scavenge DPPH*- radical and Fe³⁺ - reducing power. No correlation between SOD activity of extracts and concentration of flavonoids was found (p?≥?0.2).Thus, transformation has led to the alteration in flavonoid accumulation and antioxidant activity in A. vulgaris “hairy” roots. Transgenic roots with high-antioxidant properties can be selected after A. rhizogenes-mediated transformation.     

Hairy root culture of <Emphasis Type="Italic">Picrorhiza kurroa</Emphasis> Royle ex Benth.: a promising approach for the production of picrotin and picrotoxinin

Picrorhiza kurroa Royle ex Benth. is an endangered plant producing various compounds of medicinal importance. Hairy roots of P. kurroa were obtained following cocultivation of shoot tip explants with Agrobacterium rhizogenes strains A 4 and PAT 405. Bacterial strain A 4 appeared to be better than the strain PAT 405 in terms of both growth of respective hairy root cultures and secondary metabolite production. The optimal growth of both the hairy root cultures occurred on half-strength semisolid medium with 3% sucrose. Picrotin and picrotoxinin from the roots of wild type field grown plants were compared with 8-week-old hairy root cultures induced by the A 4 and PAT 405 strains of A. rhizogenes. Picrotin and picrotoxinin content were evaluated in hairy root cultures as well as roots of field grown plant of P. kurroa. In terms of the production of picrotin and picrotoxinin, the A 4 induced hairy roots appeared to be a better performer than the PAT 405 induced hairy root cultures. The picrotin and picrotoxinin content was highest in 8-week-old A 4 induced hairy roots (8.8 μg/g DW and 47.1 μg/g DW, respectively). Rapid growth of the hairy roots of P. kurroa with in vitro secondary metabolite production potential may offer an attractive alternative to the exploitation of this endangered plant species.     

Potential of the genetically transformed root cultures of Plumbago europaea for biomass and plumbagin production

Plumbago europaea L. is the main source of plumbagin which is a well-known pharmacological active compound. In this investigation, genetically transformed roots of P. europaea were obtained by improving some factors affecting the efficiency of Agrobacterium rhizoigenes-mediated transformation such as explant type, A. rhizoigenes strain, bacterial infection period, co-cultivation period and acetosyringone concentration. The leaf, hypocotyl and stem explants from in vitro grown plantlets were infected with bacterial strains (A4, ATCC15834, MSU440 and A13). The highest transformation rate of 69.3% was achieved after 7–9 days by inoculating A. rhizogenes MSU440 strain onto the 3-week-old stem explants followed by a co-cultivation period of 2 days on a medium containing 100 μM acetosyringone. To investigate the existence of the rolB gene, polymerase chain reaction was carried out using specific primers. Effects of growth media (MS, 1/2 MS, MS-B5 and ½ MS-B5), different sucrose concentrations and illumination on biomass production and plumbagin biosynthesis in P. europaea hairy root cultures were analyzed using stem explants after infection with MSU440 strain. ½ MS-B5 liquid medium containing 30 g L⁻¹ sucrose incubated in the dark resulted in the efficient biomass production of transformed hairy roots (12.5 g fresh weight, 1.8 g dry weight) with 3.2 mg g⁻¹ DW plumbagin accumulation. This procedure provides a framework for large-scale cultivation of hairy roots for plumbagin production. This is the first report describing the establishment of P. europaea hairy root culture with special emphasis on plumbagin production.     

Establishment of Gymnema sylvestre hairy root cultures for the production of gymnemic acid

Gymnema sylvestre is an important medicinal plant that bears bioactive compound namely gymnemic acid. In the present study, G. sylvestre was transformed by Agrobacterium rhizogenes. Seedling explants namely roots, stems, hypocotyls, cotyledonary nodal segments, cotyledons and young leaves were inoculated with A. rhizogenes strain KCTC 2703. Transformed (hairy) roots were induced from cotyledons and leaf explants. Six transgenic clones of hairy roots were established and confirmed by polymerase chain reaction (PCR) and RT-PCR using rolC specific primers. Hairy roots cultured using MS liquid medium supplemented with 3 % sucrose showed highest accumulation of biomass (97.63 g l^?1 FM and 10.92 g l^?1 DM) at 25 days, whereas highest accumulation of gymnemic acid content (11.30 mg g^?1 DM) was observed at 20 days. Nearly 9.4-fold increment of biomass was evident in suspension cultures at 25 days of culture and hairy root biomass produced in suspension cultures possessed 4.7-fold higher gymnemic acid content when compared with the untransformed control roots. MS-based liquid medium was superior for the growth of hairy roots and production of gymnemic acid compared with other culture media evaluated (B5, NN and N6), with MS-based liquid medium supplemented with 3 % sucrose was optimal for secondary metabolite production. The current results showed great potentiality of hairy root cultures for the production of gymnemic acid.     

Hairy root culture of <Emphasis Type="Italic">Plumbago indica</Emphasis> as a potential source for plumbagin

Hairy roots of Plumbago indica were established at high frequency (90 %) by infecting leaf explants with Agrobacterium rhizogenes strain ATCC 15834. The axenic root cultures were established under darkness in hormone-free liquid Murashige and Skoog medium containing 3 % sucrose. The highest plumbagin content was found to accumulate in roots at their exponential phase of growth. A low pH (4.6) and a low concentration of sucrose (1 %) were beneficial for root growth in darkness, while pH 5.6 and 3 % sucrose under continuous irradiance enhanced plumbagin accumulation in roots up to 7.8 mg g⁻¹(d.m.). Direct shoot regeneration from hairy root culture was also achieved under continuous irradiance, thus indicated an easy way of obtaining transformed P. indica plants.     

Induction of <Emphasis Type="Italic">Gentiana cruciata</Emphasis> hairy roots and their secondary metabolites

Gentiana cruciata L. (cross gentian) is a medicinal and ornamental plant. The root extracts of this species are known to exhibit many curative properties. The natural Gentiana populations are exposed to great danger because of their uncontrolled usage. In this study, hairy roots from Gentiana cruciata L. stem and leaf explants belonging to three different clones were induced by inoculation with four different Agrobacterium rhizogenes wild strains namely A4, 15834, 8196 and R1000. Induction of the root transformation was significantly dependent on the explant type used. On the other hand, the genotype and bacterial strain had no significant effect on hairy root formation. Hairy root formation percentages of the explants varied between 5.6–33.3% in the stem explants, and between 0.0–6.7% in the leaf explants. Transformations of the hairy roots were confirmed by PCR using rolC specific primers, and revealed the absence of contaminating A. rhizogenes with virC primers. Total of twelve hairy root clones were obtained, and their secondary metabolite content was also analyzed by HPLC. Quantitative results exhibited that gentiopicroside was the most abundant compound in all root samples. Furthermore, metabolites such as loganic acid, swertiamarin, and sweroside were also identified and quantified in the samples.     

The effect of yeast extract and methyl jasmonate on rosmarinic acid accumulation in <Emphasis Type="Italic">Coleus blumei</Emphasis> hairy roots

The leaves of axenically grown Coleus blumei were inoculated with Agrobacterium rhizogenes strain A4 and hairy root were established. PCR and Southern hybridization analysis confirmed transgenic nature of hairy root clones. Cultures of normal roots, induced by α-naphthaleneacetic acid on leaf explants, and hairy roots were evaluated for growth and rosmarinic acid content. Significantly better growth and up to 2.8 higher amount of rosmarinic acid was detected by HPLC analysis in hairy root clones. Methyl jasmonate stimulated rosmarinic acid accumulation in 6 out of 11 tested clones, while yeast extract induced RA accumulation in two and diminished it in 5 out of 11 tested hairy root clones.     

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 10 days after the inoculation with A. rhizogenes ATCC 15834 and 45 s 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 14.3 g L⁻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.     

Production of transgenic Aralia elata regenerated from Agrobacterium rhizogenes-mediated transformed roots

Transgenic hairy roots were induced from petiole and root segments of in vitro plant Aralia elata, a medicinal woody shrub, after co-cultivation with A. rhizogenes ATCC 15834. The percentage of putative hairy root induction from root segments was higher (26.7%) than petiole explants (10.0%). Hairy roots showed active production of lateral roots with vigorous elongation. Transgenic plants were regenerated from hairy roots via somatic embryogenesis. These plants had wrinkled leaves, short petioles and numerous lateral hairy roots. The RT-PCR analysis showed the expression of rol A, B, C, D, aux 1 and 2 genes differed between the transgenic lines. Endogenous IAA level was higher in transgenic than non-transgenic plants. Conclusively, transgenic hairy roots were developed for first time in A. elata and the transgenic hairy root lines showed distinct morphological growth pattern and gene expression.     

Hernandulcin in hairy root cultures of Lippia dulcis

The hairy root culture of Lippia dulcis Trev., Verbenaceae, was established by transformation with Agrobacterium rhizogenes A4. The transformed roots grew well in Murashige and Skoog medium containing 2% sucrose. The roots turned light green when they were cultured under 16 h/day light. The green hairy roots produced the sweet sesquiterpene hernandulcin (ca. 0.25 mg/g dry wt) together with 20 other mono- and sesquiterpenes, while no terpenes were detected in the nontransformed root cultures. The growth and hernandulcin production in the hairy root cultures were influenced by the addition of auxins to the medium. The addition of a low concentration of chitosan (0.2 – 10.0 mg / l) enhanced the production of hernandulcin 5-fold.Abbreviations Cht chitosan - IAA indole-3-acetic acid - NAA 1-naphthaleneacetic acid - 2,4-D 2,4-dichlorophenoxyacetic acid - MS Murashige and Skoog(1962)     

银杏毛状根褐化机制的初步研究

该实验依据现有发根农杆菌侵染法,以银杏无菌苗叶片为外植体,对农杆菌诱导银杏毛状根的实验操作进行优化,并观察记录银杏毛状根继代生长情况,测定继代不同天数的毛状根组织内多酚含量、多酚合成相关酶基因和POD基因相对表达量,以探讨银杏毛状根褐化的生理机制。结果显示:(1)银杏农杆菌侵染最适预培养时间为3d、菌种为R1601、共培养基为White、侵染时间为30min,优化侵染操作后的银杏毛状根诱导率可达72.36%;银杏毛状根继代生长的最适IBA浓度为0.5mg/L。(2)银杏毛状根初始发根时颜色为乳白色,多在伤口处簇生,伸长生长时无向地性,不分叶正背面都能在伤口处诱导出根,且在除菌培养20d时毛状根长可达0.4～0.8cm;继代培养25d时毛状根为白色,继代35d时部分毛状根从根部至中部开始转黄,继代45d时大部分毛状根由白转黄,根部至中部开始有褐化出现。(3)继代35d时毛状根的PAL、C4 H、4CL和POD基因表达量分别为25d的68%、44%、51%、52%;继代45d时PAL、C4 H、4CL、POD基因表达量分别为35d的19%、75%、86%、105%。(4)继代35d、45d的银杏毛状根总酚类含量分别为17.14和13.78mg/g。研究发现,PAL基因表达量变化与其下游的两个关键酶基因(C4 H、4CL)表达量变化不一致,且PAL基因表达量的大幅降低并未对下游C4 H、4CL基因表达有明显影响,说明PAL基因表达量变化对其下游合成速率影响不大,可能不是银杏毛状根苯丙氨酸代谢途径的限速酶;而C4 H与4CL基因表达量变化趋势高度一致,说明C4H应是该途径中的限速酶之一;推测继代前期可能是由PPO作为关键酶参与毛状根褐化反应,导致后期毛状根生长受抑制,POD基因表达水平随之上升,POD参与褐化反应。     

Agrobacterium rhizogenes-mediated transformation of scented geranium

A method is described for producing genetically transformed plants from explants of three scentedPelargonium spp. Transgenic hairy root lines were developed fromPelargonium spp leaf explants and microcuttings after inoculation withAgrobacterium rhizogenes strains derived from the agropine A4 strain. Hairy root lines grew prolifically on growth regulator-free medium. Transgenic shoots were regenerated from hairy roots and the plants have been successfully transferred to soil. The phenotype of regenerated plants has been characterized as having abundant root development, more leaves and internodes than the controls, short internodes and highly branched roots and aerial parts. Southern blot analyses have confirmed the transgenic nature of these plants.     

Production of some benzylisoquinoline alkaloids in Papaver armeniacum L. hairy root cultures elicited with salicylic acid and methyl jasmonate

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.

     

The influence of Agrobacterium rhizogenes on induction of hairy roots and ß-carboline alkaloids production in Tribulus terrestris L.

We have developed an efficient transformation system for Tribulus terrestris L., an important medicinal plant, using Agrobacterium rhizogenes strains AR15834 and GMI9534 to generate hairy roots. Hairy roots were formed directly from the cut edges of leaf explants 10–14 days after inoculation with the Agrobacterium with highest frequency transformation being 49 %, which was achieved using Agrobacterium rhizogenes AR15834 on hormone-free MS medium after 28 days inoculation. PCR analysis showed that rolB genes of Ri plasmid of A. rhizogenes were integrated and expressed into the genome of transformed hairy roots. Isolated transgenic hairy roots grew rapidly on MS medium supplemented with indole-3-butyric acid. They showed characteristics of transformed roots such as fast growth and high lateral branching in comparison with untransformed roots. Isolated control and transgenic hairy roots grown in liquid medium containing IBA were analyzed to detect ß-carboline alkaloids by High Performance Thin Layer Chromatograghy (HPTLC). Harmine content was estimated to be 1.7 μg g⁻¹ of the dried weight of transgenic hairy root cultures at the end of 50 days of culturing. The transformed roots induced by AR15834 strain, spontaneously, dedifferentiated as callus on MS medium without hormone. Optimum callus induction and shoot regeneration of transformed roots in vitro was achieved on MS medium containing 0.4 mg L⁻¹ naphthaleneacetic acid and 2 mg L⁻¹ 6-benzylaminopurine (BAP) after 50 days. The main objective of this investigation was to establish hairy roots in this plant by using A. rhizogenes to synthesize secondary products at levels comparable to the wild-type roots.     

Shoot and root culture of Hypericum perforatum L. transformed with Agrobacterium rhizogenes A4M70GUS

Hairy root cultures of Hypericum perforatum were obtained following inoculation of aseptically germinated seedlings with A. rhizogenes strain A4M70GUS. Effect of sucrose on the growth and biomass production of hairy root cultures was investigated. Hairy root cultures spontaneously regenerated shoots buds from which a number of shoot culture clones was established. Transformed shoot cultures exhibited good shoot multiplication, elongation and rooting on a hormone-free woody plant medium. Plants regenerated from hairy roots were similar in appearance to the normal, nontransformed plants.     

Hairy roots of Brassica napus: I. Applied glutamine overcomes the effect of phosphinothricin treatment

Summary Hairy roots of Brassica napus (rape cv. Giant) were produced by cocultivating leaf and cotyledon explants with Agrobacterium rhizogenes strain A4T. The hairy roots grew prolifically on solid and in liquid media. Incorporation of ammonium sulphate or phosphinothricin (PPT) into the media reduced growth. PPT treatment reduced glutamine synthetase (GS) activity and increased the ammonia content of the hairy roots. We have found that PPT treatment also induces a loss of glutamine from the roots and this may influence root growth. To test this we grew hairy roots in a liquid medium containing 10 mM glutamine. This glutamine treatment overcame the PPT induced suppression of growth but also significantly increased GS activity, reduced ammonia accumulation and increased the levels of glutamate and asparagine.     

Optimization of induction and culture conditions and tropane alkaloid production in hairy roots of <Emphasis Type="Italic">Anisodus acutangulus</Emphasis>

In this study, an efficient transformation system for the medicinal plant Anisodus acutangulus was successfully developed and optimized using Agrobacterium rhizogenes. Three bacterial strains, A4, R1601, and modified C58C1 and three explant types, leaf blade, petiole, and stem, were examined. The highest transformation efficiency of 94.44% was achieved using strain C58C1 with stem explants. Over 20 independent hairy root lines were successfully established with strain C58C1 using stem explants, all of which contained the ro/B and ro/C genes as confirmed by polymerase chain reaction (PCR). Out of four media compositions, the liquid 1/2 MS medium was found the most suitable for hairy root growth. The maximum biomass of one hairy root line increased up to 80 times in liquid 1/2 MS medium after a 30 day culture period. Different hairy root lines displayed a varied capacity for tropane alkaloid production and the best hairy root line (T4) from the C58C1-stem combination produced up to 10.21 mg/g (dw) of hyoscyamine, which was about 1.5-fold higher than in the wild type plants. To our knowledge, this is the first report to demonstrate the production of tropane alkaloids in hairy roots of A. acutangulus.     

Hairy roots induced by Agrobacterium rhizogenes and production of regenerative plants in hairy root cultures in maize

Hairy roots of maize were induced by infecting 15-d calli with Agrobacterium rhizogenes. The hairy roots cultured in hormone-free media showed the vigorous growth and typical hairy root features. The regenerated plants were produced from hairy roots in MS media supplemented with 1.6 mg/L ZT and 0.4 mg/L NAA. The PCR-Southern hybridization demonstrated that T-DNA had been integrated into the chromosome of regenerated plants.