Genetic transformation of <Emphasis Type="Italic">Harpagophytum procumbens</Emphasis> by <Emphasis Type="Italic">Agrobacterium rhizogenes</Emphasis>: iridoid and phenylethanoid glycoside accumulation in hairy root cultures

A genetic transformation method using Agrobacterium rhizogenes was developed for Harpagophytum procumbens. The influence of three factors on hairy root formation was tested: bacterial strains (A4 and ATCC 15834), various types of explants and acetosyringone (AS) (200 μM). The highest frequency of transformation (over 50% of explants forming roots at the infected sites after 6 weeks of culture on Lloyd and McCown (WP) medium) was achieved using a combination of nodal stem explants and A. rhizogenes strain A4. The addition of 200 μM AS to root induction medium was found to enhance hairy root induction but its effect varied depending on bacterial strain and explant type. Three of the most vigorously growing hairy root clones of H. procumbens were chosen and analyzed for accumulation of iridoid and phenylethanoid glycosides. The transgenic nature of these root clones was confirmed by PCR amplification; they were positive for rolB and rolC genes. Harpagoside, verbascoside and isoverbascoside were identified by HPLC and LC–ESI-MS as the major compounds from all analyzed hairy root clones. The Hp-3 root clone showed the higher harpagoside content (0.32 mg g⁻¹ dry wt.) compared with other analyzed transformed and non-tuberized untransformed roots of H. procumbens. However, the level of the compound in the hairy root clone was lower than that detected in a sample of commercially available root tubers of H. procumbens. The Hp-3 root clone also produced high amounts of verbascoside and isoverbascoside (8.12 mg g⁻¹ dry wt. and 9.97 mg g⁻¹ dry wt., respectively) comparable to those found in root tubers.     

Hairy root cultures of Rehmannia glutinosa and production of iridoid and phenylethanoid glycosides

Hairy root lines through the infection of Agrobacterium rhizogenes strain (A4) were established from shoot tips and leaves of Rehmannia glutinosa Libosch. Ten lines of hairy roots were selected on the basis of biomass increase in half-strength Gamborg medium (¹/₂ B5). Transgenic status of the roots was confirmed by polymerase chain reaction using rolB and rolC specific primers. Iridoid glycosides (catalposide, loganin, aucubin and catalpol) and phenylethanoid glycosides (verbascoside and isoverbascoside) identified using HPLC?CESI?CMS, and their contents were compared with untransformed root culture and roots of 1-year-old field-grown plants of R. glutinosa by RP-HPLC. The growth and production of secondary metabolites in ten hairy root lines varied considerably as to the media. Woody plant (WP) medium displayed higher growth in terms of fresh (FW) and dry weights (DW) compared to ¹/₂ B5 medium. High-yielding hairy root lines produced higher amounts of loganin, catalposide, verbascoside and isoverbascoside in comparison to the untransformed root culture and roots of 1-year-old field-grown plants. The highest amounts of catalposide and loganin in transformed roots were 4.45?mg?g^?1 DW (RS-2 hairy root line) and 4.66?mg?g^?1 DW (RS-1 hairy root line), respectively. Aucubin and catalpol were detected in some lines in trace amounts. The highest amounts of verbascoside (16.9?mg?g^?1 DW) and isoverbascoside (3.46?mg?g^?1 DW) were achieved in RS-2 root line. The contents of catalposide, verbascoside and isoverbascoside in high-producing lines were several times higher than in untransformed root culture and roots of R. glutinosa plants grown in soil. Loganin and aucubin could not be detected in roots of field-grown plants. However, the levels of catalpol were much lower in the in vitro roots.     

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.     

Nickel tolerance and hyperaccumulation in shoot cultures regenerated from hairy root cultures of <Emphasis Type="Italic">Alyssum murale</Emphasis> Waldst et Kit

Shoot cultures of nickel hyperaccumulating Alyssum murale were established from epicotyl explants of seedlings aseptically germinated on hormone-free MS medium. They were further maintained on media with 0–0.92 μM kinetin. Optimal shoot multiplication was at 0.46 μM kinetin. Inoculation by shoot wounding was performed with overnight suspension of A. rhizogenes A4M70GUS which contains GUS gene cointegrated in pRiA4. After 30 days hairy roots were produced at the wounding site in 31 explant (25% out of 124). Hairy roots were excised and further propagated on hormone-free medium as separate clones. In the first passage clones 3 and 6 could be distinguished by fast growth and spontaneous shoot regeneration. In other clones (12, 23 and 25) shoot regeneration required presence of cytokinins. The five shoot culture clones regenerated from hairy roots were further cultured on media with 0.46 μM kinetin. These shoots were characterized by good elongation and lateral shoot branching, short internodes, minute slightly curled leaves and well developed plagiotropic root system spreading over the surface of media. Thus all plants regenerated from hairy root cultures manifested the characteristic Ri syndrome phenotype. They all had a strong positive GUS reaction. PCR analysis confirmed presence of uidA sequence from the gus construct. They were also tolerant to nickel accumulating up to 24,700 μg g⁻¹ dry weight.     

Agrobacterium rhizogenes-mediated transformation of Picrorhiza kurroa Royle ex Benth.: establishment and selection of superior hairy root clone

A protocol for induction and establishment of Agrobacterium rhizogenes-mediated hairy root cultures of Picrorhiza kurroa was developed through optimization of the explant type and the most suitable bacterial strain. The infection of leaf explants with the LBA9402 strain resulted in the emergence of hairy roots at 66.7% relative transformation frequency. Nine independent, opine and TL-positive hairy root clones were studied for their growth and specific glycoside (i.e., kutkoside and picroside I) productivities at different growth phases. Biosynthetic potentials for the commercially desirable active constituents have been expressed by all the tested hairy root clones, although distinct inter-clonal variations could be noted in terms of their quantity. The yield potentials of the 14-P clone, both in terms of biomass as well as individual glycoside contents (i.e., kutkoside and picroside I), superseded that of all other hairy root clones along with the non-transformed, in vitro-grown control roots of P. kurroa. The present communication reports the first successful establishment, maintenance, growth and selection of superior hairy root clone of Picrorhiza kurroa with desired phyto-molecule production potential, which can serve as an effective substitute to its roots and thereby prevent the indiscriminate up-rooting and exploitation of this commercially important, endangered medicinal plant species. CIMAP Publication No.: 2007-28J     

A novel life cycle arising from leaf segments in plants regenerated from horseradish hairy roots

Summary Horseradish (Armoracia rusticana) hairy root clones were established from hairy roots which were transformed with the Ri plasmid in Agrobacterium rhizogenes 15834. The transformed plants, which were regenerated from hairy root clones, had thicker roots with extensive lateral branches and thicker stems, and grew faster compared with non-transformed horseradish plants. Small sections of leaves of the transformed plants generated adventitious roots in phytohormone-free G (modified Gamborg's) medium. Root proliferation was followed by adventitious shoot formation and plant regeneration. Approximately twenty plants were regenerated per square centimeter of leaf. The transformed plants were easily transferable from sterile conditions to soil. When leaf segments of the transformed plants were cultured in a liquid fertilizer under non-sterile conditions, adventitious roots were generated at the cut ends of the leaves. Adventitious shoots were generated at the boundary between the leaf and the adventitious roots and developed into complete plants. This novel life cycle arising from leaf segments is a unique property of the transformed plants derived from hairy root clones.     

<Emphasis Type="Italic">Agrobacterium rhizogenes</Emphasis> mediated transformation of <Emphasis Type="Italic">Scutellaria baicalensis</Emphasis> and production of flavonoids in hairy roots

Using different explants of in vitro seed grown Scutellaria baicalensis Georgi plantlets, hairy roots were induced following inoculation of Agrobacterium rhizogenes strains A₄GUS, R1000 LBA 9402 and ATCC11325. The A₄GUS proved to be more competent than other strains and the highest transformation rates were observed in cotyledonary leaf explant (42.6 %). The transformed roots appeared after 15–20 d of incubation on hormone free Murashige and Skoog medium. Growth of hairy roots was assessed on the basis of total root elongation, lateral root density and biomass accumulation. Maximum growth rate was recorded in root:medium ratio 1:100 (m/v). Hairy root lines were further established in Gamborg B₅ medium and the biomass increase was maximum from 15 to 30 d. PCR, Southern hybridization and RT-PCR confirmed integration and expression of left and right termini-linked Ri T-DNA fragment of the Ri plasmid from A₄GUS into the genome of Scutellaria baicalensis hairy roots. GUS assay was also performed for further integration and expression. All the clones showed higher growth rate them non-transformed root and accumulated considerable amounts of the root-specific flavonoids. Baicalin content was 14.1–30.0 % of dry root mass which was significantly higher then that of control field grown roots (18 %). The wogonin content varies from 0.08 to 0.18 % among the hairy root clones which was also higher than in non-transformed roots (0.07 %).     

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.     

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.     

Assessment of conditions affecting <Emphasis Type="Italic">Agrobacterium</Emphasis> <Emphasis Type="Italic">rhizogenes</Emphasis>-mediated transformation of soybean

Agrobacterium rhizogenes-mediated transformation has become a powerful tool for studying gene function and root biology due to its quick and simple methodology. This transformation method is particularly suitable for those plants, including legumes, whose transformation using Agrobacterium tumefaciens has been challenging. Although there are some reports on A. rhizogenes-mediated transformation of legumes to produce ‘composite’ plants, conditions influencing A. rhizogenes-mediated transformation of soybean [Glycine max (L.) Merr.] have not been yet fully investigated. To better understand A. rhizogenes-mediated root transformation in soybean, we have evaluated the impact of genotype, plant age for infection, bacterial inoculating concentration, inoculation temperature, and other factors on transformation of soybean. The results have shown that there are significant differences among soybean genotypes in their susceptibility to A. rhizogenes. Soybean cv. Zigongdongdou is the most susceptible to A. rhizogenes strain K599 among 10 genotypes tested. The effects of seedling age have been evaluated, and 1-day-old plantlets are found to be optimal for hairy root induction. There are no significant differences in hairy root induction for bacterial suspension from OD₆₀₀ = 0.2 to OD₆₀₀ = 1.2. Under 16 h photoperiod, hairy roots can be induced both at 23°C/20°C and 28°C/25°C, but not at 33°C/30°C as day/night temperature regimes. Using this transformation protocol, almost 100% of the composite plants formed hairy roots within 2 weeks, and based on GUS histochemical analysis, 94.2% transformation frequency is obtained. Transgene integration has been also confirmed by Southern blot analysis. D. Cao and W. Hou contributed equally to this work.     

Genetic transformation of <Emphasis Type="Italic">Ruta graveolens</Emphasis> L. by <Emphasis Type="Italic">Agrobacterium rhizogenes</Emphasis>: hairy root cultures a promising approach for production of coumarins and furanocoumarins

Ruta graveolens L. is a source of pharmacologically active compounds such as coumarins, furanocoumarins and furoquinolone alkaloids. Hypocotyls, callus and shoots of R. graveolens were inoculated with bacteria from two Agrobacterium rhizogenes strains. Hairy root cultures were established after inoculation of hypocotyls with wild A. rhizogenes strain LBA 9402. The transgenic nature of the regenerated tissue was confirmed by PCR amplification. Coumarins, furanocoumarins and alkaloids present in the hairy root tissue were identified by GC and GC-MS and compared with those present in in vitro shoot cultures. The level of pinnarin and rutacultin, bergapten, isopimpinelin and xanthotoxin was approximately twofold higher in hairy root than in shoot cultures. Two additional coumarins: osthole and osthenol, never been found in R. graveolens, were identified in hairy root tissue. Besides coumarins, alkaloids were identified: dictamnine, skimmianine, kokusaginine, rybalinine and an isomer of rybalinine. The levels of nearly all coumarins and alkaloids in hairy roots cultured in the darkness were higher than those accumulated under a photoperiod mode.     

Influence of <Emphasis Type="Italic">Agrobacterium rhizogenes</Emphasis> on induction of hairy roots and benzylisoquinoline alkaloids production in Persian poppy (<Emphasis Type="Italic">Papaver bracteatum</Emphasis> Lindl.): preliminary report

Persian poppy (Papaver bracteatum Lindl.) is an important medicinal plant and source of the opium alkaloids codeine, morphine and thebaine. Transgenic root cultures of P. bracteatum Lindl. are well-defined model systems to investigate the molecular and metabolic regulation of benzylisoquinoline alkaloid biosynthesis. Agrobacterium rhizogenes was able to produce hairy roots on wounded Persian poppy seedlings. Excised shoots from 7-day-old Persian poppy were co-cultivated with the A. rhizogenes strain R15834 carrying the pBI121 binary vector. All media, except for the co-cultivation medium, included 40 mg l⁻¹ paromomycin to select for pBI121 transformants and 200 mg l⁻¹ cefotaxime to eliminate the Agrobacterium. Eight weeks after infection, paromomycin-resistant roots appeared on 45–50% of explants maintained on hormone-free medium. Isolated hairy roots were propagated in liquid medium containing 1.0 mg l⁻¹ 1-naphthaleneacetic acid to promote rapid growth. Also, callus induction and shoot regeneration of transformed Calli in vitro was achieved on B5 medium containing 1.0 mg l⁻¹ 1-naphthaleneacetic acid. Detection of the neomycin phosphotransferase gene and GUS histochemical localization confirmed the integrative transformation of root cultures. This is the first study to illustrate useful protocol to introduce foreign genes into transgenic Persian poppy hairy root cultures using A. rhizogenes strain R15834.     

Induction of hairy roots and plant regeneration from the medicinal plant <Emphasis Type="Italic">Pogostemon Cablin</Emphasis>

An efficient transformation system for the medicinal and aromatic plant, Pogostemon cablin Benth was developed by using agropine-type Agrobacterium rhizogenes ATCC15834. Hairy roots formed directly from the cut edges of leaf explants or via callus stage 8 days after inoculation with the bacterium. The highest frequency of leaf explant transformation by Agrobacterium rhizogenes ATCC15834 was about 80% after infection for 25 days. Hairy roots grew rapidly on plant growth regulators (PGRs)-free Murashige and Skoog (MS) or 6,7-V medium and had characteristics of transformed roots such as fast growth and high lateral branching. The PCR amplification showed that rol genes of Ri plasmid of A. rhizogenes were integrated and expressed into the genome of transformed hairy roots. The hairy root line, PL6, grew very slowly in the first 8 days, then grew very quickly between day 8 and day 24. The optimum medium for callus induction of hairy roots consisted of 2.0 mg l⁻¹ benzyladenine (BA) and 0.1 mg/l α-naphthaleneacetic acid (NAA); while optimum medium for adventitious shoot regeneration from these cultures consisted of 0.1 mg l⁻¹ BA and 0.1 mg l⁻¹ NAA. Adventitious shoots could be rooted on 1/2MS. Southern blot analysis confirmed that rol genes of TL-DNA of Ri plasmid was integrated with at least three copies into the genome of hairy roots- regenerated P. cablin plants. The results presented provide a solid foundation for production of patchouli essential oil from hairy roots or its regenerated plants and also provide possibilities for utilization of artifical polyploidization or chemical mutation of hairy roots for improving germplasm and breeding of a new cultivar of P. cablin.     

Genetic transformation of Rhamnus fallax and hairy roots as a source of anthraquinones

Hairy roots of Rhamnus fallax Boiss. were induced using Agrobacterium rhizogenes strain A4M70GUS. The culture established on Woody plant media (WPM) showed a typical hairy root phenotype: rapid growth, reduced apical dominance and root plagiotropism. Seven clones of R. fallax were selected on the basis of their differences in colour and the root branching. The growth of hairy root culture, measured through gain in fresh mass, was done under 16-h photoperiod or in the dark. An increase in anthraquinone (AQ) content was obtained in clones with yellow and less branched roots, like clone 1 [16.43 mg g⁻¹(d.m.)] and clone 7 [14.21 mg g⁻¹(d.m.)], compared with other analysed transformed and non-transformed tissue. This study presents the first report of successful transformation of any species from family Rhamnaceae by A. rhizogenes and analysis of AQ production in transformed tissue.     

Transformation through agroinfection on decapitated shoot apex of field-growing <Emphasis Type="Italic">Phyllanthus amarus</Emphasis>

A simple and easy transformation strategy was accomplished on field growing plants of Phyllanthus amarus, an anti-hepatitis B drug plant. Infection of Agrobacterium rhizogenes strains A₄M70GUS and ATCC 15834 on decapitated shoots of field growing P. amarus induced hairy roots and crown gall, respectively. Infection with A₄M70GUS yielded a mean of 23.2 roots from 40% plants in 40-day period. The crown gall induced on 30% plants after infection with ATCC 15834 grew to 5–10 mm in diameter. The roots and crown galls established in vitro on Murashige and Skoog (MS) basal medium grew well. The hairy roots yielded fivefold (6.91 g) biomass in half-strength MS liquid medium to that of the adventitious roots derived from internode explants in MS medium with 8.0 μM α-naphthaleneacetic acid (1.39 g). Histochemical assay and PCR analysis using the primers of uidA coding region confirmed the hairy roots induced by A₄M70GUS. The crown galls induced by ATCC 15834 were confirmed by PCR analysis using rolB gene primers. The protocol enables an easy and early accomplishment of hairy roots.     

重金属超富集植物东南景天毛状根的诱导

为建立重金属超富集植物东南景天(Sedum alfredii)的毛状根诱导体系,采用发根农杆菌(Agrobacterium rhizogenes)A4侵染叶片,研究了预培养时间、侵染时间和共培养时间对毛状根诱导率的影响。结果表明,东南景天叶片外植体的预培养时间为48 h、农杆菌侵染时间为6 min、共培养时间为48 h是适宜的毛状根诱导时间,毛状根的诱导率可达85%。PCR检测表明诱导的毛状根中存在rol B基因片段。这是东南景天首次建立用发根农杆菌诱导毛状根体系。     

Sporamin-mediated resistance to beet cyst nematodes (Heterodera schachtii Schm.) is dependent on trypsin inhibitory activity in sugar beet (Beta vulgaris L.) hairy roots

Sporamin, a sweet potato tuberous storage protein, is a Kunitz-type trypsin inhibitor. Its capability of conferring insect-resistance on transgenic tobacco and cauliflower has been confirmed. To test its potential as an anti-feedant for the beet cyst nematode (Heterodera schachtii Schm.), the sporamin gene SpTI-1 was introduced into sugar beet (Beta vulgaris L.) by Agrobacterium rhizogenes-mediated transformation. Twelve different hairy root clones expressing sporamin were selected for studying nematode development. Of these, 8 hairy root clones were found to show significant efficiency in inhibiting the growth and development of the female nematodes whereas 4 root clones did not show any inhibitory effects even though the SpTI-1 gene was regularly expressed in all of the tested hairy roots as revealed by northern and western analyses. Inhibition of nematode development correlated with trypsin inhibitor activity but not with the amount of sporamin expressed in hairy roots. These data demonstrate that the trypsin inhibitor activity is the critical factor for inhibiting growth and development of cyst nematodes in sugar beet hairy roots expressing the sporamin gene. Hence, the sweet potato sporamin can be used as a new and effective anti-feedant for controlling cyst nematodes offering an alternative strategy for establishing nematode resistance in crops.     

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.