Polyacetylenes accumulation in <Emphasis Type="Italic">Ambrosia maritima</Emphasis> hairy root and cell cultures after elicitation with methyl jasmonate

Three lines of hairy root culture of Ambrosia maritima induced by Agrobacterium rhizogenes ATCC15834 were established. Thiarubrine A, thiarubrine A epoxide, thiarubrine A diol and their precursor pentayneene were produced by the hairy roots after elicitation with methyl jasmonate, the common signal molecule in the plant defense and development. Thiarubrine A diol was the main form detected in the medium. Maximum yield was achieved when the 13-day-old hairy root cultures were exposed to 40 M methyl jasmonate for 72 h. Callus and cell suspension cultures were established and maintained on Murashige and Skoog medium supplied with -naphthylacetic acid (NAA) and kinetin. When the cell suspension cultures were elicited with methyl jasmonate, pentayneene was the only polyacetylene produced. The yield of pentayneene in hairy root cultures was much higher (9.6 times) than that of cell suspension cultures.     

Expression of transferred genes during hairy root development in pea

Root border cell development and expression of reporter genes were evaluated in transgenic pea hairy roots. Successful induction of hairy roots in pea is conditioned by bacterial strain and plant genotype, as well as by developmental and environmental factors. Morphological changes sometimes occur when hairy roots are transferred from infected plants to tissue culture media, but such changes are confined to specific clones. Expression of reporter genes under the control of promoters from bean (Phaseolus vulgaris L.) stress genes encoding phenylalanine ammonia lyase and chalcone synthase were evaluated. Expression patterns vary between hairy roots taken directly from infected plants, and those grown in culture; most hairy roots taken from infected plants exhibit expression throughout all tissues, whereas expression in cultured hairy roots is most often localized to specific tissues. Patterns of expression that occur during different stages of hairy root development are very similar to those observed in transgenic plants expressing the same fusion genes. Border cell separation and release in hairy roots is normal, and expression of glucuronidase in border cells of some transgenic roots resulted in development of bright blue single cells. Cultured hairy roots should provide a very useful model for studying the effect of defined changes in root border cells on microbial associations with roots of this important legume.Abbreviations YEM yeast extract-mannitol - GUS glucuronidase - PAL phenylalanine ammonium lyase - CHS chalcone syntase     

Eugenol from normal and transformed root cultures of Coluria geoides

Transformed root cultures of Coluria geoides Ledeb. were established with the use of Agrobacterium rhizogenes LBA 9402. Both normal and transformed root cultures were investigated for their growth and yield of eugenol. Normal roots were grown in B5 medium-supplemented with 0.2 mg l^-1 of kinetin and 0.2 mg l^-1 of 1-naphthaleneacetic acid (NAA). Hairy roots grew well in hormone-free B5 medium. Both hairy roots and normal roots produced glycosidic bound eugenol. as with the roots of intact plants, eugenol was the main component of the total essential oils obtained from hairy root and normal root cultures. The yield of eugenol from normal roots was 0.1–0.25% of the dry wt. and depended on the development stage of the culture. Yield of eugenol from hairy roots was 0.08–0.1% of the dry wt. NAA modified the hairy root morphology and influenced the yield of eugenol.Abbreviations NAA 1-naphthaleneacetic acid     

Comparative evaluation of modified bioreactors for enhancement of growth and secondary metabolite biosynthesis usingPanax ginseng hairy roots

Hairy root cultures have demonstrated great promise in terms of their biosynthetic capability toward the production of secondary metabolites, but continue to constitute a major challenge with regard to large-scale cultures. In order to assess the possibility of conducting mass production of biomass, and the extraction of useful metabolites fromPanax ginseng. P. ginseng hairy roots, transformed byRhizobium rhizogenes KCTC 2744, were used in bioreactors of different types and sizes. The most effective mass production of hairy roots was achieved in several differently sized air bubble bioreactors compared to all other bioreactor types. Hairy root growth was enhanced by aeration, and the production increased with increasing aeration rate in a 1 L bioreactor culture. It was determined that the hairy root growth rate could be substantially enhanced by increases in the aeration rate upto 0.5 wm, but at aeration rates above 0.5 wm, only slight promotions in growth rates were observed. In 20 L air bubble bioreactors, with a variety of inoculum sizes, the hairy roots exhibited the most robust growth rates with an inoculum size of 0.1% (w/v), within the range 0.1 to 0.7% (w/v). The specific growth rates of the hairy roots decreased with increases in the inoculum size.     

Hairy root transformation in alfalfa (Medicago sativa L.)

Summary The widely cultivated forage legume alfalfa (Medicago sativa L.) was transformed with the agropine type Agrobacterium rhizogenes NCPPB 1855. Sterile root and callus cultures were derived from tumorous hairy roots which were easily obtained independent of the plant variety or genotype. Plant regeneration, via somatic embryogenesis, was achieved only when a selected alfalfa line, characterized by high regenerative capability, was utilized. Genetic transformation was confirmed by the presence of agropine and T-DNA. Phenotypic alterations, mainly affecting the root system, were observed in transformed plants. The possibility that T-DNA-induced variations could be useful in the improvement of M. sativa is discussed.Research work was partially supported by Progetto Strategico Agrobiotecnologia C.N.R., Italy     

An interchangeable system of hairy root and cell suspension cultures ofCatharanthus roseus for indole alkaloid production

Hairy roots ofCatharanthus roseus obtained by co-cultivation of hypocotyl segments withAgrobacterium rhizogenes, and cultured in SH (Schenk and Hildebrandt) basal medium, formed two types of calli when subcultured in SH medium with 1 mg/1 -naphthaleneacetic acid and 0.1 mg/l kinetin. One of them, a compact callus, when re-subcultured in SH basal medium gave rise to hairy roots again. A rhizogenic cell suspension culture was established from this type of callus. When cultured in SH medium with growth regulators, the rhizogenic callus produced catharanthine at a level of 41% of the level in the initial hairy roots. Upon transfer to SH basal medium, regenerated hairy roots produced this alkaloid at the original level of 1.5 mg/g dry wt. Using this cell/hairy root interchange system a new management system for hairy root culture in bioreactors has been devised and examined involving production of biomass in the form of a cell suspension in medium supplemented with growth regulators, and catharanthine production by hairy roots regenerated from these cells in medium without growth regulators.Abbreviations NAA -naphthaleneacetic acid - SH Schenk and Hildebrandt - SHNK SH medium + 1 mg 1^–1 NAA + 0.1 mg 1^–1 kinetin     

Rosmarinic acid production by <Emphasis Type="Italic">Coleus forskohlii</Emphasis>hairy root cultures

Coleus forskohlii hairy root cultures were found to produce forskolin and rosmarinic acid (RA) as the main metabolites. The growth and RA production by C. forskohlii hairy root cultures in various liquid media were examined. The hairy root cultures showed good growth in hormone-free Murashige and Skoog medium containing 3% (w/v) sucrose (MS medium), and Gamborg B5 medium containing 2% (w/v) sucrose (B5 medium). RA yield reached 4.0 mg (MS medium) and 4.4 mg (B5 medium) after 5 weeks of culture in a 100 ml flask containing 20 ml of each medium. Hairy root growth and RA were also investigated after treatment with various concentrations of yeast extract (YE), salicylic acid (SA) and methyl jasmonic acid (MJA). RA production in a 100 ml flask containing 20 ml B5 medium reached 5.4 mg (1.9 times more than control) with treatment of 0.01 or 1% (w/v) YE, 5.5 mg (2.0 times more than control) with treatment of 0.1 mM SA, and the maximum RA content with 9.5 mg per flask (3.4 times more than control) was obtained in the hairy roots treated with 0.1 mM MJA. These results suggest that MJA is an effective elicitor for production of RA in C. forskohlii hairy root cultures.     

Rose-scented geranium (<Emphasis Type="Italic">Pelargonium</Emphasis> sp.) generated by <Emphasis Type="Italic">Agrobacterium rhizogenes</Emphasis> mediated Ri-insertion for improved essential oil quality

Transgenic plants of rose-scented geranium (Pelargonium graveolens cv. Hemanti) have been produced from Agrobacterium rhizogenes (strains A₄ and LBA9402) mediated hairy root cultures. Amongst the explants tested, leaves were most responsive followed by the petioles and internodal segments, respectively. The A₄ strain performed better for all the three explants both in terms of frequency of response and time requirement for hairy root induction. Transgenic shoots could be obtained by spontaneous regeneration without intervening callus phase amongst 16% and 12% root lines of A₄ and LBA 9402 origin, respectively, or they were induced in 29% and 22% hairy root lines of A₄ and LBA9402 origin, respectively, with different hormonal supplementation. These transgenic plants showed 30% survival as against 90% of their control under the confined environment of glasshouse. The transgenic plants were of similar morphotype having increased branching, higher number of leaves with increased dentations, short and round stature, highly branched root system and absence of leaf wrinkling. These transgenic plants showed opine positive results even after 5 months of their transfer to the glasshouse. The essential oil compositions of 81% of these transgenics were qualitatively similar to that of the wild type parent. However, two transgenic plants (LZ-3 and 14TG) showed increase in concentrations of geraniol and geranyl esters signifying improved oil quality with respect to the citronellol:geraniol ratio. These two oils having better olfactory value represent an improvement over that of the wild type parent from the commercial point of view.     

Enhanced secondary metabolite (tanshinone) production of <Emphasis Type="Italic">Salvia miltiorrhiza</Emphasis> hairy roots in a novel root–bacteria coculture process

Salvia miltiorrhiza Bunge (Lamiaceae) hairy root cultures were inoculated (at 0.02 and 0.2% v/v) and co-cultured with Bacillus cereus bacteria. The root biomass growth was inhibited significantly by the bacteria inoculated to the root culture on the first day (day 0) but not by the bacteria inoculated on days 14 or 21 (in a 28-day overall period). On the other hand, the growth of the bacteria in the hairy root culture was also strongly inhibited by the hairy roots, partially because of the antibacterial activity of the secondary compounds produced by the roots. Most interestingly, the tanshinone production was promoted by the inoculation of bacteria at any of these days but more significantly by an earlier bacteria inoculation. With 0.2% bacteria inoculated on day 0, for example, the total tanshinone content of roots was increased by more than 12-fold (from 0.20 to 2.67 mg g⁻¹ dry weight), and the volumetric tanshinone yield increased by more than sixfold (from 1.40 to 10.4 mg l⁻¹). The tanshinone production was also stimulated by bacterial water extract and bacterial culture supernatant but less significantly than by the inoculation of live bacteria. The results suggest that the stimulation of tanshinone production by live bacteria in the root cultures may be attributed to the elicitor compounds originating from the bacteria, and the hairy root–bacteria coculture may be an effective strategy for improving secondary metabolite production in plant tissue cultures.     

Production of diosgenin by hairy root cultures ofTrigonella foenum-graecum L.

Hairy root cultures ofTrigonella foenum-graecum L. were established withAgrobacterium rhizogenes strain A₄. The hairy roots produce diosgenin, an important spirostanol for the semi-synthesis of steroid hormones. Fourteen different liquid media were investigated. The fastest growth was obtained in McCown's woody plant (WP) medium supplemented with 3% sucrose; the highest diosgenin content was observed in half-strength WP medium with 1% sucrose (0.040% dry weight), which represents almost twice the amount detected in the 8-month-old non-transformed roots (0.024%). A time-course study in WP liquid media supplemented with 3% sucrose was undertaken. In these conditions, 17 g diosgenin/g fresh weight were produced. The influence of cholesterol, medium pH and chitosan on diosgenin production was tested. The addition of 40 mg/l chitosan elevated the diosgenin content to three times that found in non-elicited hairy roots.Abbreviations MS Murashige and Skoog (1962) medium - WP McCown's woody plant medium     

Production of steroidal alkaloids by hairy roots of Solanum aviculare and the effect of gibberellic acid

Cultures of Solanum aviculare hairy roots were established after transformation with Agrobacterium rhizogenes A4. High levels of steroidal alkaloids measured as solasodine equivalents were produced in shake-flasks and bioreactor, even though relatively low concentrations are found in roots in vivo. In shake flasks the maximum alkaloid yield was 32 mg g^-1 dry weight; in a 3-1 air-driven bioreactor the yield was 29 mg g^-1. These yields represent a 5-fold increase over previous reports for in vitro production, and are comparable with levels found in the aerial parts of intact S. aviculare plants. Production of steroidal alkaloids was growth-associated. High sugar levels at stationary phase and insensitivity to increased levels of medium components suggest that root cultures were limited by oxygen mass-transfer. In Petri-dish culture with and without exogenous gibberellic acid, root length and number of root tips increased exponentially; growth proceeded with a constant length per root tip of about 35 mm. Addition of gibberellic acid enhanced growth but reduced the specific steroidal-alkaloid level. Taking into account both growth and alkaloid yield, accumulation of steroidal alkaloids was improved by about 40% at gibberellic-acid concentrations of 10 and 100 g l^-1.     

Agrobacterium-mediated transformation of Artemisia absinthium L. (wormwood) and production of secondary metabolites

Hairy roots were obtained after infection of Artemisia absinthium shoots with Agrobacterium rhizogenes strains 1855 and LBA 9402. The susceptibility to hairy root transformation varied between plant genotypes and bacterial strains. Hairy roots showed macroscopic differences from control root cultures. Southern blot hybridization confirmed the integration of T-DNA from both p1855 and pBin19, while polymerase chain reaction analysis indicated the presence of the neomycin phosphotransferase gene in the hairy root genome. Subcultured transformed root lines grew well in selective B5 agar-solidified medium containing kanamycin or rifampicin and without hormones. Shake-flask experiments with fast-growing root lines showed that 40 g l^–1 was the best sucrose concentration for biomass production, yielding a 463-fold increase in dry weight after 28 days of culture. Great differences were found in the profiles of the essential oils isolated from normal and hairy roots. Gas chromatography/mass spectrometry analysis showed the oil produced by transformed cultures to be a mixture of 50 compounds with only one major component representing 37% of the oil content. Received: 19 March 1996 / Revision received: 15 July 1996 / Accepted: 13 December 1996     

Assessment of the essential oil composition of Tornabenea annua, Tornabenea insularis and Tornabenea tenuissima fruits from Cape Verde Islands

The essential oils of Tornabenea annua, Tornabenea insularis and Tornabenea tenuissima herbarium or in vivo fruits, collected in Fogo, Santiago, Santo Antão, São Nicolau and São Vicente Islands, from Cape Verde archipelago, or from plants grown in Lisbon, Portugal, were isolated by hydrodistillation and analysed by GC and GC–MS. The yellowish oils were obtained in variable average yields, lower in herbarium samples [0.05% (v/w)] and higher from in vivo samples [1.3% (v/w)]. Whereas T. annua fruits' oils were all dominated by myristicin (92–100%), most of the T. insularis fruit samples' oils were elemicin rich (82–90%). No clear information could be obtained for T. tenuissima fruits' oils as each of the samples gave different chemical composition. Cluster analysis of the essential oil composition from the fruits' samples studied, confirmed these chemical differences.     

Transformation of <Emphasis Type="Italic">Saussurea medusa</Emphasis> for hairy roots and jaceosidin production

Axenically grown Saussurea medusa plantlets were inoculated with four Agrobacterium rhizogenes strains, and hairy root lines were established with A. rhizogenes strain R1601 in N6 medium. PCR and Southern hybridization confirmed integration of the T-DNA fragment of the Ri plasmid from A. rhizogenes into the genome of S. medusa hairy roots. In N6 medium, maximum biomass of the hairy root cultures was achieved [8 g (dry weight) per liter; growth ratio 35-fold] after 21 days of culture. The amount of jaceosidin extracted from the hairy root cultures was 46 mg/l (production ratio of 37-fold) after 27 days of culture. The maximum jaceosidin content obtained using N6 medium was higher than that obtained with Modified White, MS or B5 medium. In N6 medium, the tip segments were more efficient for hairy root growth and jaceosidin production than the middle and basal regions of the root.Abbreviations AS Acetosyringone - BA Benzyladenine - cef Cefotaxime sodium - DW Dry weight - FW Fresh weight - HPLC High-performance liquid chromatography - IAA Indole-3-acetic acid - km Kanamycin - NAA -Naphthaleneacetic acid - SDS Sodium dodecyl sulfate     

Composition and biological activities of essential oils from Limnophila geoffrayi Bonati

Essential oils from the dried aerial parts of Limnophila geoffrayi Bonati were obtained by water-distillation. d-Pulegone (27.14%), perillaldehyde (19.13%) and limonene (9.00%) were characterized as the major constituents using gas chromatography-mass spectrometry analysis. The antimicrobial activities of the essential oils and their major components were evaluated against microorganisms encountered normally in contaminated cosmetic products, using the agar- and broth-dilution methods. Their insecticidal activities against the Oriental fruit fly Bactrocera dorsalis (Hendel) were tested using a bioassay with impregnated filter paper. The results showed that the essential oils possessed high antimicrobial activity, with minimum inhibitory concentrations ranging from 0.03 to 0.2% per unit volume (v/v). Strong insecticidal activity as a fumigant was also observed at an oil dose of 5 μl/disc, with a 94% mortality. Perillaldehyde was the most active compound among the main components of these essential oils.     

Ecophysiological characterization of carnivorous plant roots: Oxygen fluxes,respiration, and water exudation

Various ecophysiological investigations on carnivorous plants in wet soils are presented. Radial oxygen loss from roots of Droseraceae to an anoxic medium was relatively low 0.02 – 0.07 mol(O₂) m^{– 2} s^–1 in the apical zone, while values of about one order of magnitude greater were found in both Sarracenia rubra roots and Genlisea violacea traps. Aerobic respiration rates were in the range of 1.6 – 5.6 mol kg^–1 (f.m.) s^–1 for apical root segments of seven carnivorous plant species and 0.4 – 1.1 mol kg^–1 (f.m.) s^–1 for Genlisea traps. The rate of anaerobic fermentation in roots of two Drosera species was only 5 – 14 % of the aerobic respiration. Neither 0.2 mM NaN₃ nor 0.5 mM KCN influenced respiration rate of roots and traps. In all species, the proportion of cyanide-resistant respiration was high and amounted to 65 – 89 % of the total value. Mean rates of water exudation from excised roots of 12 species ranged between 0.4 – 336 mm 3 kg^–1 (f.m.) s^–1 with the highest values being found in the Droseraceae. Exudation from roots was insensitive to respiration inhibitors. No significant difference was found between exudation rates from roots growing in situ in anoxic soil and those kept in an aerated aquatic medium. Carnivorous plant roots appear to be physiologically very active and well adapted to endure permanent soil anoxia.     

Accumulation of essential oils by Agrobacterium tumefaciens-transformed shoot cultures of Pimpinella anisum

Axenic transformed shoot cultures of Pimpinella anisum (anise) were established following inoculation of plant stems with the nopaline strain T37 of Agrobacterium tumefaciens. The stable incorporation of T-DNA in the transformed tissues was demonstrated by polymerase chain reaction. Total essential oil accumulated by transformed shoot cultures grown under continuous light was found to be 18% lower (per unit fresh weight of tissue) than that produced by untransformed shoot cultures incubated under similar conditions, but more than 89% lower than the yield of oil from the intact plant. The relative amounts of the principal components of the essential oil of the transformed shoot cultures, namely geraniol, -bisabolene, trans-pseudoisoeugenol-2-methylbutyrate and transanethole, were similar to those present in the parent plant, but significantly different from those of the untransformed shoot cultures.Abbreviations T-DNA transfer-DNA - MS Murashige and Skoog medium - 2,4-D 2,4-dichlorophenoxyacetic acid - TY tryptone and yeast extract medium - t_D doubling time - GC-MS gas chromatography coupled with mass spectrometry - FID flame ionisation detector - PCR polymerase chain reaction - TE Tris-HCl, EDTA buffer - TBE Tris, borate, EDTA buffer     

Initiation of and solasodine production in hairy root cultures of Solanum mauritianum Scop.

Initiation and establishment of hairy root cultures from leaf or seedling hypocotyl explants of Solanum mauritianum Scop., using six strains of Agrobacterium rhizogenes was attempted. Success was only achieved following hypocotyl inoculation with strain LBA 9402. Transformation frequency was very low, with only one instance out of a possible 90 being recorded. Resultant hairy root cultures grew rapidly and could be maintained using a Murashige and Skoog (1962) medium supplemented with 0.1 g L^–1 myo-inositol and 3% sucrose, either as a solid or liquid culture. Under these conditions, the roots had a solasodine content of 126 g g^–1 DW. Lower levels of solasodine and decreased root growth rates were recorded when the medium strength was reduced by half or 3% glucose substituted for the 3% sucrose.Abbreviations MS Murashige and Skoog's (1962) medium     

Stimulation of saponin production in <Emphasis Type="Italic">Panax ginseng</Emphasis> hairy roots by two oligosaccharides from <Emphasis Type="Italic">Paris polyphylla</Emphasis> var. <Emphasis Type="Italic">yunnanensis</Emphasis>

Two oligosaccharides, a heptasaccharide (HS) and an octasaccharide (OS), isolated from Paris polyphylla var. yunnanensis, stimulated the growth and saponin accumulation of Panax ginseng hairy roots at 5–30 mg l⁻¹. HS and OS at 30 mg l⁻¹, fed separately to hairy root cultures at 10 days post-inoculation, increased the root biomass dry weight by more than 70% to ∼20 g l⁻¹ from 13 g l⁻¹ and the total saponin content of roots by more than 1-fold to ∼3.5% from 1.6% (w/w). The results suggest that the two oligosaccharides may have plant growth-regulatory activity in plant tissue cultures.     

Enhancement of growth and secondary metabolite biosynthesis: Effect of elicitors derived from plants and insects

Plant-derived natural products have been and will continue to be valuable sources. Elicitors have been employed to modify cell metabolism in order to enhance the productivity of useful metabolites in plant cell/tissue cultures. In this study, several elicitors were used to improve the productivity of useful metabolites and to reduce culture time for archiving high concentration inP. ginseng hairy root cultures. The addition of chitosan, chitosan oligosaccharide and alginate oligosaccharide to the culture ofP. ginseng hairy roots caused growth to be inhibited with the increase in elicitor concentration. The usage of the chitosan elicitor andd-glucosamine caused a slight decrease in hairy root growth, whereas total ginseng saponin accumulated slightly with the increase in elicitor concentration. When gel beads were added to the culture medium at the initial period, hairy root growth was enhanced. The maximum growth was 1.35 times higher than that of the control at 1% (w/v). Total ginseng saponin content decreased due to the addition of alginate beads. This would result in consistent diffusion of lower levels of calcium ions during the culture period that promotes biomass growth.