Production of valepotriates by hairy root cultures of Centranthus ruber DC

Hairy root cultures of Centranthus ruber DC. were established by infection of sterile plantlets with Agrobacterium rhizogenes, strain R1601. The transformed roots were grown in 12 different, hormone-free liquid media, and valtrate, isovaltrate, 7-desisovaleroyl-7-acetylvaltrate, 7-homovaltrate, didrovaltrate and isovaleroxyhydroxydidrovaltrate were quantified by high performance liquid chromatography. The highest overall valepotriate content (3.0% dry wt) was observed in half-strength Gamborg B5 medium supplemented with 3% sucrose. This concentration is very similar to that found in the roots of parent plants grown in the field. The use of N,N-dimethylmorpholinium iodide, a plant bioregulator, was very detrimental to the hairy root growth and to the valepotriate production. The hairy roots cultured in half strength Gamborg B5 liquid medium supplemented with 3% sucrose for 45 days produced over 31 mg/g dry wt valepotriates.Abbreviations MS Murashige and Skoog medium (Murashige and Skoog 1962) - B5 Gamborg B5 medium (Gamborg 1970) - WP McCown's woody plant medium (Lloyd and McCown 1980) - H Heller's medium (Heller 1953) - 1/4 B5-7 quarter strength B5 + 7% sucrose - DMI N,N-dimethylmorpholmium iodide - VAL valtrate - IVAL isovaltrate - DIA-VAL 7-desisovaleroyl-7-acetylvaltrate - HVAL 7-homovaltrate - DI didrovaltrate - IVHD isovaleroxyhydroxydidrovaltrate (Fig. 1) - NMR nuclear magnetic resonance spectroscopy - HPLC high performance liquid chromatography - Ac acetyl - IV isovaleryl - IV-IV -isovaleryloxy-isovaleryl - MV -methyl-valeryl     

High-yield production of valepotriates by hairy root cultures of Valeriana officnalis L. var. sambucifolia Mikan

Summary Hairy root cultures of Valeriana officinalis var. sambucifolia were established by infection of sterile plantlets with Agrobacterium rhizogenes strain R1601 The transformed roots were grown in 10 different, hormone-free liquid media and the isovaltrate, valtrate, didrovaltrate, isovaleroxyhydroxydidrovaltrate content was quantified by HPLC. Valepotriates were entirely retained inside the root tissues. The highest overall valepotriate content (10.3 % dry wt), 4 times the amount found in the roots of 9-month-old nontransformed plants, was observed in half strength Gamborg B5 medium supplemented with 2 % sucrose. The hairy roots cultured in Murashige and Skoog liquid medium supplemented with 2 % sucrose for 50 days produced over 44 mg/g dry wt valepotriates.Abbreviations MS Murashige and Skoog medium (Murashige and Skoog 1962) - B5 Gamborg B5 medium (Gamborg 1970) - WP McCown's woody plant medium (Lloyd and McCown 1980) - 1/2 MS-2 half strength MS+2 % sucrose - 1/4 B5-2 quarter strength B5+2% sucrose - MS-7 full strength MS+7% sucrose - YMB Yeast mannitol broth (Hooykaas et al. 1977) - IVAL isovaltrate - VAL valtrate - IVHD isovaleroxyhydroxydidrovaltrate - DI didrovaltrate (Fig. 1)     

Characteristics of growth and tropane alkaloid production in Hyoscyamus albus hairy roots transformed with Agrobacterium rhizogenes A4

Summary Hairy root culture of Hyoscyamus albus was established by transformation with Agrobacterium rhizogenes strain A4. The growth and production of five tropane alkaloids were investigated under various culture conditions. Among the four basal culture media tested, Woody Plant medium was the best for growth of the hairy roots, but a high amount of tropane alkaloids was obtained with Gamborg's B5 medium. Sucrose concentration in B5 medium had little effect on the growth, while 3% sucrose was suitable for the alkaloid production. Addition of KNO₃ to Woody Plant medium affected the growth, whereas the alkaloid content was not markedly improved. Supplement of some metal ions to B5 medium stimulated the alkaloid production. In particular, Cu²⁺ remarkably enhanced both the growth and the alkaloid yield. The hairy roots cultured under 16 h/day light survived for more than 32 days compared with those cultured in the dark.Abbreviations EDTA ethylenediaminetetraacetic acid - HPLC high performance liquid chromatography - MeOH methanol - MS medium Murashige and Skoog medium - WP medium McCown's Woody Plant medium - B5 medium Gamborg B5 medium - wt weight     

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     

Establishment of fast-growing callus and root cultures of Cephalotaxus harringtonia

Summary Callus cultures were established from Cephalotaxus harringtonia (Japanese plumyew) stem expiants cultured on Murashige and Skoog medium supplemented with 4.5 M 2,4-dichlorophenoxyacetic acid and 0.05 M 6-furfurylaminopurine. The inclusion of 4.9 M 6-(,-dimethylallylamino) purine as the sole hormone significantly increased the growth rate of the callus. Organogenesis giving rise to both shoots and roots occurred upon transfer of the callus onto a hormonefree medium. Vitrification was common on all regenerated shoots cultured on Gelrite-containing medium. Regenerated roots were excised and established in McCown's woody plant medium. Doubling the phosphate and nitrate levels in the medium increased the growth of these root cultures.Abbreviations MS Murashige and Skoog basal medium - B5 Gamborg's B5 basal salt medium - WP McCown's woody plant basal salt medium - 2,4-D 2,4-dichlorophenoxyacetic acid - Kinetin 6-furfurylamino-purine - 2iP 6-(,-dimethylallylamino) purine - IBA indole-3-butyric acid     

Lobeline production by hairy root culture of Lobelia inflata L.

Hairy roots were obtained following inoculation of the stems of Lobelia inflata L. with Agrobacterium rhizogenes strain ATCC 15834. These hairy roots contained agropine and mannopine. In addition, lobeline was detected by HPLC and confirmed by mass spectrometry. Various media were tested for the growth of hairy roots as well as for the content of lobeline in hairy roots. The growth rate of hairy roots cultured in Nitsch and Nitsch's medium was approximately one third of those cultured in other media. The lobeline content of hairy roots (18–54 g/g dry weight) cultured in these media was the same order of magnitude compared with that of roots of L. inflata (24 g/g dry weight) cultivated in pots. The hairy roots cultured in Nitsch and Nitsch's medium were morphologically different from those cultured in other media.Abbreviations MS medium Murashige and Skoog's medium - 1/2 MS medium one-half strength of the standard Murashige and Skoog's medium - B5 medium Gamborg's B5 medium - NN medium Nitsch and Nitsch's medium - FW fresh weight - DW dry weight     

In vitro propagation of the leguminous tree Swartzia madagascariensis

Shoot induction frequency for the leguminous tree Swartzia madagascariensis Desv. was higher on MS and WP media than on B5. Explants incubated on media solidified with agar produced more shoots with a lower tendency to hyperhydricity than explants on agarose or Gelrite media. Maximum shoot induction was obtained with an agar-solidified MS medium containing 2.2 M benzyladenine (37 shoots/explant). Shoots rooted after transfer to half-strength MS medium supplemented with 26.8 M naphthaleneacetic acid.Abbreviations BA benzyladenine - IBA indolebutyric acid - NAA naphthaleneacetic acid - WP(M) woody plant (medium)     

Somatic embryogenesis and plant development from immature zygotic embryos of seedless grapes (Vitis vinifera L.)

Summary Somatic embryo formation occurred from immature zygotic embryos within ovules of stenospermocarpic seedless grapes (Vitis vinifera L.), when cultured for two months on liquid Emershad/Ramming medium. Somatic embryos continued to proliferate after excision and transfer to Emershad/Ramming medium supplemented with 1 M benzylaminopurine and 0.65% TC agar. Plant development from somatic embryos was influenced by genotype, medium, phase (liquid, agar), stage (torpedo, mature) and their interactions. Optimal plant development occurred on Woody Plant Medium supplemented with 1.5% sucrose + 1 M benzylaminopurine + 0.3% activated charcoal and 0.65% TC agar.Abbreviations ABA abscisic acid - BAP benzylaminopurine - ER Emershad/Ramming - GLM general linear model - MGLH multivariate general linear hypothesis - MS Murashige/Skoog - NaClO sodium hypochlorite - TC tissue culture - WP woody plant     

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     

In vitro regeneration of Alnus cremastogyne Burk from epicotyl explants

Summary Multiple shoots were grown from seedling explants of Alnus cremastogyne Burk by a two-stage culture procedure: initiation on WP medium supplemented with 2–8 M benzylammopurine(BAP) for 6 weeks, thereafter 3 weeks of subculture(shoot multiplication) on the same medium with 1 M BAP. A 5–9 fold multiplication rate was achieved. Type and concentration of sugar used in the multiplication medium were shown to be critical factors for both multiple shoot induction and bud elongation, the optima being 87.5mM glucose and 87.5mM sucrose respectively. After transfer to half-strength WP media either containing indolebutyric acid (IBA) or lacking plant growth regulator, almost all the shoots rooted. However, high rhizogenesis could be achieved only with shoots cultured in rooting medium containing 87.5mM sucrose or 175mM glucose, and shoots from multiplication media containing 87.5mM sucrose. Survival of the plantlets following transfer to vermiculite was 100%.Abbreviations BAP 6-benzylaminopurine - 2iP N⁶-(²-isopentenyl)adenine - kinetin 6-furfurylaminopurine - zeatin trans-6-(4-hydroxy-3-methylbut-2-enyl)aminopurine - IBA indol-3-butyric acid - WPM Woody plant medium (Lloyd and McCown, 1981)     

<Emphasis Type="Italic">Agrobacterium rhizogenes</Emphasis>-mediated DNA transfer to<Emphasis Type="Italic"> Aesculu</Emphasis>s<Emphasis Type="Italic"> hippocastanum</Emphasis> L. and the regeneration of transformed plants

Hairy roots were induced from androgenic embryos of horse chestnut (Aesculus hippocastanum L.) by infection with Agrobacterium rhizogenes strain A4GUS. Single roots were selected according to their morphology in the absence of antibiotic or herbicide resistance markers. Seventy-one putative transformed hairy root lines from independent transformation events were established. Regeneration was induced in MS liquid medium supplemented with 30 6-benzylaminopurine (BA), and the regenerants were multiplied on MS solid medium containing 10 M BA. Following elongation on MS medium supplemented with 1 M BA and 500 mg/l polyvinylpyrrolidone, the shoots were subjected to a root-inducing treatment. Stable integration of TL-DNA within the horse chestnut genome was confirmed by Southern hybridization. The copy number of transgenes was estimated to be from two to four.Communicated by E.D. Earle     

Growth and steroidal saponin production in hairy root cultures of Solanum aculeatissimum

Summary Hairy root cultures of Solanum aculeatissimum were established by trans-formation using Agrobacterium rhizogenes strain 15834. Root growth and production of steroidal saponin were investigated under various culture conditions. Transformed roots grew better in Gamborg's B5 medium containing 3 % sucrose under continuous light than in the dark. Also, the roots turned light green when cultured under continuous light. Green hairy roots produced aculeatiside A (6.71mg ·) L^–1 and aculeatiside B (6.39mg · L^–1) after 8 weeks of culture, while no steroidal saponin was detected in hairy roots cultured in the dark. Of the three culture media tested, Gamborg's B5 medium was superior for growth and steroidal saponin production. Growth and steroidal saponin production were enhanced when 100g · L^–1 auxin except for 2,4-D was added to the medium. The addition of 2,4-D inhibited growth. Production of steroidal saponin was highest with NAA. Transformed roots used in this experiment were confirmed that hairy roots examined contain both TL-DNA and TR-DNA region of Ri plasmid by PCR amplification analysis of DNA.Abbreviations MS medium Murashige and Skoog's medium (1962) - B5 medium Gamborg's B5 medium (1968) - LS medium Linsmaier and Skoog's medium(1965) - HPLC High performance liquid chromatography - NAA -Naphthaleneacetic acid - IAA Indole-3-acetic acid - 2,4-D 2,4-Dichlorophenoxyacetic acid - PCR polymerase chain reaction     

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)     

Traits of Panax ginseng hairy roots after cold storage and cryopreservation

Summary Panax ginseng hairy root cultures were established by infecting petiole segments with Agrobacterium rhizogenes strain 15834. Hairy root segments including root tips placed onto phytohormone-free 1/2 Murashige and Skoog solid medium and stored at 4 °C in the dark for 4 months, resumed elongation when the temperature was raised to 25 °C in the dark. For cryopreservation, a vitrification method was applied. Root tips precultured with 0.1 mg/l 2,4-D for 3 days and dehydrated with PVS2 solution for 8 minutes prior to immersion into liquid nitrogen had a survival rate of 60 % and could regenerate. The hairy roots regenerated from cryopreserved root tips grew well and showed the same ginsenoside productivity and patterns as those of the control hairy roots cultured continuously at 25 °C. The conservation of T-DNAs in the regenerated hairy roots was proved by PCR analysis.Abbreviations 1/2 MS a half strength Murashige and Skoog (1962) - B5 Gamborg B5 (Gamborg et al. 1968) - WP woody plant (Lloyd and McCown 1980) - RC root culture (Thomas and Davey 1982) - RCI root culture medium containing 100 mg/l myoinositol - HF phytohormone-free - IAA indole-3-acetic acid - IBA indole-3-butyric acid - 2,4-D 2,4-dichlorophenoxyacetic acid - TIBA 2,3,5-triiodobenzoic acid - PCR polymerase chain reaction - PVS2 plant vitrification solution 2 (Sakai et al., 1990) - FDA fluorecein diacetate     

Elicitation of thiophene production by cultured hairy roots of Tagetes patula

Hairy roots of Tagetes patula were initiated by infecting the seedlings with Agrobacterium rhizogenes. The hairy roots were grown in liquid medium MS (Murashige and Skoog 1962) supplemented with sucrose. The roots were treated with three different elicitors obtained from mycelial culture of Aspergillus niger, Rhizopus oligosporus and Pencillium notatum. Accumulation of biomass and the production of thiophenes were studied over a period of six weeks in culture. The HPLC separation profile of the thiophenes indicated the presence of several structurally different thiophenes. α-terthienyl being predominent. Maximum production of thiophene was recorded at the end of the fourth week in culture with a content of 0.138 % (w/w on dry weight basis). Treatment of hairy roots with mycelial extract of Aspergillus niger (1.5 % v/v) elicited an increase in thiophene content by 1.6 folds over the control.     

Callus induction from protoplasts of V. unguiculata,V. sublobata and V. mungo

Summary Protoplasts were isolated from hypocotyl of V. mungo (L.) Hepper or hypocotyl-derived callus of V. sublobata (Phaseolus sublobata Roxb.) and V. unguiculata (L.) Walp (syn. V. sinensis (L.) Saviex Hassk) using an enzyme solution comprising Cellulase 2.5%, Macerozyme, Hemicellulase and Driselase each at a 0.5% level in 0.5 M sorbitol. Isolated protoplasts were cultured in Murashige and Skoog's (1962) basal liquid medium supplemented with BA, NAA, 2,4-D (1 mg/l each) and sucrose (14%). After four weeks, protoplast colonies were transferred to the same medium with a reduced level of sucrose (7%). Colonies proliferated into actively growing calli. Further attempts to regenerate plants from such calli were not successful. However, protoclones of V. unguiculata differentiated roots on auxin/cytokinin supplemented media. Alternative methods for shoot differentiation from protoplastderived cultures were tried by the use of Agrobacterium tumefaciens shooter strains pGV 2215 or pGV 2298 or wild type strain B6S3.     

Hairy root cultures of Anethum graveolens (dill): establishment, growth, time-course study of their essential oil and its comparison with parent plant oils

Transformed root cultures of Anethum graveolens were induced by inoculation of aseptically grown seedlings with Agrobacterium rhizogenes carrying plasmid pRi 1855. The main component of the essential oils from the fruits and from the roots of the parent plant was carvone, whereas -phellandrene and apiole were dominant in the oil from, respectively, the aerial parts and the hairy roots. The essential oils from the fruits, aerial parts and roots of the parent plant were at 2%, 0.3% and 0.06% (v/w), respectively, but only 0.02% (v/w) in the hairy root cultures. Growth of the hairy root cultures reached 600 mg dry wt/50 ml medium after 50 days. The essential oil composition did not change significantly during their growth.     

Indigo production in hairy root cultures of Polygonum tinctorium Lour.

The transformed root culture of Polygonum tinctorium Lour. was established by infecting leaf explants with Agrobacterium rhizogenes A4. These cultures were examined for their growth and indigo content under various culture conditions. Among the four different culture media tested, SH medium showed the highest yield for root growth (28 mg dry wt/30 ml) and indigo production (152 g/dry wt). In SH medium, 30 g sucrose l^–1, 2500 mg KNO₃ l^–1, 300 mg NH₄H₂PO₄ l^–1 were the best conditions for indigo production at pH 5.7. The production of indigo in hairy roots slightly increased with the addition of 200 mg chitosan l^–1 (186 g/dry wt) and 20 U pectinase l^–1 (181 g/dry wt).