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)     

An unusual root tip formation in hairy root culture of Hyoscyamus muticus

Summary Hairy root cultures of Hyoscyamus muticus were established using Agrobacterium rhizogenes ATCC 15834. In one out of 8 clones established, an unusual root tip formation was observed after transfer of cultures from half-strength Murashige and Skoog (1962) to White's medium (1939). This phenomenon was associated with the production of a fine brownish cell suspension culture. Hairy root development resumed after transfer of the root tips from White to half-strength Murashige and Skoog medium. After plating the isolated brownish cells on hormone-free half-strength Murashige and Skoog or White solid medium, callus proliferation was observed, and then redifferentiation of hairy roots occurred. The polymerase chain reaction analysis of the H. muticus hairy root (clone Z2) revealed that only the tl region of the T-DNA was integrated. The growth and the production of five tropane alkaloids by this clone were examined.Abbreviations PCR Polymerase Chain Reaction - MS medium Murashige and Skoog Medium - 1/2 MS medium half-strength MS medium - WP medium Woody Plant medium - RC medium Root Culture medium - WH medium White medium - HPLC High Performance Liquid Chromatography - wt. weight     

Shikonin production and secretion by hairy root cultures of Lithospermum erythrorhizon

Summary Hairy root cultures of Lithospermum erythrorhizon were established by transformation of in vitro grown shoots with Agrobacterium rhizogenes 15834. Hairy roots cultured on Murashige and Skoog solid medium did not produce any red pigments. However, the hairy roots cultured in Root Culture solid or liquid media produced a large amount of red pigments, which were released to the medium. The addition of adsorbents to the culture medium stimulated shikonin production by ca. 3-fold. Using this method an air-lift fermenter system was established, equipped with a XAD-2 column, which continuously produced ca. 5 mg/day of shikonin during a period of more than 220 days.     

Establishment of new axenic hairy root lines by inoculation with Agrobacterium rhizogenes

Cultured hairy root lines resulting from infection by Agrobacterium rhizogenes are known for approximately thirty plant species. We extend this range by establishing forty original dicotyledonous hairy root lines with A. rhizogenes strain A₄. Hairy roots have been cultured for at least 2–6 years on Murashige & Skoog medium. Some hairy root cultures such as Anagallis arvensis and Antirrhinum majus spontaneously regenerated whole plants.     

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     

Hairy roots formation in recalcitrant-to-transform plant Chenopodium rubrum

Susceptibility of C. rubrum to Agrobacterium-mediated transformation was demonstrated by inoculating the petioles of in vitro grown plants with A. rhizogenes strain A4M70GUS. Hairy roots were produced in 8 % of explants. They were isolated and maintained on plant growth regulator-free solid or liquid half-strength Murashige and Skoog medium for two years. Hairy root fresh mass increased 30 — 90 folds when grown in liquid medium, which was superior to solid medium, where most of the hairy roots produced calli. When these calli were grown on medium supplemented with 0.5 mg dm-3 thidiazuron, embryo-like structures were obtained. Transgenic status of long-term callus and hairy root cultures was confirmed by histochemical GUS assay, by PCR specific to the uidA, rolA&B and ags genes and by Southern hybridization.     

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     

Tropane alkaloid production by Agrobacterium rhizogenes transformed hairy root cultures of Atropa baetica Willk. (Solanaceae)

Atropa baetica hairy root cultures were induced after infecting stem segments with Agrobacterium rhizogenes strain ATCC 15834. Accumulation of the tropane alkaloids atropine and scopolamine by hairy roots cultured in half- and full-strength Murashige and Skoog (MS) medium was high, although this was not growth associated. These alkaloids were also released into both liquid media. Higher tropane alkaloids present both in hairy roots and liquid medium occurred in half MS medium, showing a clear relationship between slow growth of cultures and higher product accumulation. The pH of both nutrient media varied as culture progressed, and seemed to be associated with the release of scopolamine. GC-MS analyses showed the presence of a new compound, namely tigloylpseudotropine; moreover, 3α-isobutyryloxytropane, formerly found only in plant leaf tissue, was also identified in the hairy roots. Received: 18 August 1997 / Revision received: 30 November 1997 / Accepted: 20 January 1998     

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.     

Formation of embryogenic callus in hairy roots of pumpkin (<Emphasis Type="Italic">Cucurbita pepo</Emphasis> L.)

Summary Excised cotyledons from 8-d-old pumpkin (Cucurbita pepo L.) seedlings were inoculated with Agrobacterium rhizogenes and cultured on hormone-free Murashige and Skoog medium. At the site of inoculation, transformed hairy roots were successfully induced by using wild strains 8196 (mannopine-type) and 15834 (agropine-type). After a subsequent transfer on a solid MS medium without hormones, roots obtained by transformation with strain 15834 failed to form stable hairy root cultures, while several hairy root lines were established with strain 8196. Three hairy root lines, Cp1, Cp2, and Cp31, have spontaneously generated callus with embryo-like structures after more than 3 yr of growth on the solid medium. The callus proliferation was more frequent when the autoclaving of nutrient medium, pH 5.7, was prolonged to 30 min. Separated calluses continued to proliferate and generated embryos with abnormal morphology. The combination of indole-3-acetic acid and benzyladenine had a favorable influence on embryogenesis and organogenesis in the Cp31 callus line. The Southern analysis of Cp31 root and embryo DNA confirmed the presence of the T-DNA of Agrobacterium rhizogenes.     

In vitro Regeneration and Transformation of Blackstonia perfoliata

In vitro root culture of yellow wort (Blackstonia perfoliata (L.) Huds.) was initiated on Murashige and Skoog (MS) medium. In the presence of benzylaminopurine (BAP) numerous adventitious buds formed, which developed into shoots. Presence of indole-3-butyric acid (IBA) in media significantly decreased number of buds, but increased development of lateral roots. On hormone-free medium shoots successfully rooted and developed flowers and viable seeds that formed another generation. Shoot cultures of B. perfoliata inoculated with suspension of Agrobacterium rhizogenes strain A4M70GUS developed hairy roots at 3 weeks and they were cultured on hormone-free MS medium. Spontaneous shoot regeneration occurred in 3 clones.     

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     

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.     

Tomato (<Emphasis Type="Italic">Lycopersicon esculentum</Emphasis> cv. pera) hairy root cultures: Characterization and changes in peroxidase activity under NaCl treatment

Summary Hairy root cultures of Lycopersicon esculentum L. Mill ev. Pera were established by infection of leaf explants with Agrobacterium rhizogenes LBA 9402. The pattern of peroxidase isoenzymes in these tissues was similar to that of roots excised from tomato plants grown in hydroponic cultures. Hairy root cultures may be an appropriate system to analyze the peroxidase involvement in the response of isolated roots to salt stress, avoiding the problem of wounding or changes in hormone levels observed in roots excised from plants. The cultures of hairy roots allowed the evaluation of changes in peroxidase patterns not only in the tissue but also in the culture medium. Hairy roots were subcultured in Murashige and Skoog liquid medium with or without 100 mM NaCl to investigate the evolution of growth, total peroxidase activity of the tissue and culture medium, and changes in the peroxidase isoenzyme patterns under each condition of growth. Control cultures showed a growth index higher than those reported for other hairy root cultures, and it was even higher in the presence of 100 mM NaCl. The total peroxidase activity in the tissue was similar for control and salt-treated roots. Even when the total peroxidase activity of the medium decreased under salt treatment, NaCl induced secretion of a highly basic peroxidase and inhibition of the secretion of some acidic isoenzymes. These changes may explain the physiological role of these enzymes in the response to salt stress that we will possibly establish through a future study of the biochemical properties of those peroxidases.     

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.     

Production of ribosome-inactivating protein from hairy root cultures of Luffa cylindrica (L.) Roem.

Transformed root lines of Luffa cylindrica (L.) Roem. (Cucurbitaceae) were established by inoculation of in vitro grown plantlets with wild type Agrobacterium rhizogenes strain 1855. Cloned lines of hairy roots were tested for the presence of ribosome-inactivating proteins; crude extracts inhibited protein synthesis in a reaction mixture based on rabbit reticulocyte lysate. Inhibitory activity increased during culture period, reaching a maximum value in the stationary phase. No activity could be detected in the culture medium, nor in extracts from callus and/or suspension cultures. A ribosome-inactivating protein having specific activity of 62,100 U mg protein^–1 and a molecular mass of 26–28,000 Da was purified to homogeneity. The protein showed N-glycosidase activity on rat liver ribosomes. The results demonstrate that hairy root cultures can be successfully utilized for the in vitro production of ribosome-inactivating proteins.Abbreviations BAP benzylaminopurine - 2,4D 2,4dichlorophenoxyacetic acid - HPLC high pressure liquid chromatography - MS Murashige and Skoog - NAA naphthaleneacetic acid - NCPPB National Collection of Phytopathogenic Bacteria - Ri root-inducing - RIP ribosome-inactivating protein - UV ultra-violet - YMB yeast mannitol broth     

Hairy root culture optimization and resveratrol production from <Emphasis Type="Italic">Vitis vinifera</Emphasis> subsp. <Emphasis Type="Italic">sylvesteris</Emphasis>

Resveratrol is a polyphenolic compound produced in very low levels in grapes. To achieve high yield of resveratrol in wild grape, three Agrobacterium rhizogenes strains, Ar318, ArA4 and LBA9402, were used to induce hairy roots following infection of internodes, nodes or petioles of in vitro grown Vitis vinifera subsp. sylvesteris accessions W2 and W16, and cultivar Rasha. The effects of inoculation time, age of explants, bacterial concentration and co-cultivation times were examined on the efficiency of the production of hairy roots. Strains Ar318, ArA4 and LBA9402 all induced hairy roots in the tested genotypes, but the efficiency of ArA4 strain was higher than the other strains. The highest hairy root production was with using internodes as explants. The transformation of hairy roots lines was confirmed by PCR detection of rolB gene. Half Murashige and Skoog (MS) medium was better for biomass production compared with MS medium. HPLC analysis of resveratrol production in the hairy root cultures showed that all the genotypes produced higher amounts of resveratrol than control roots. The highest amount of resveratrol was produced from W16 internode cultures, which was 31-fold higher than that of control root. Furthermore, TLC analysis showed that treatments of hairy roots with sodium acetate and jasmonate elevated resveratrol levels both in hairy root tissue and excreted into the half MS medium. These results demonstrate that endogenous and exogenous factors can affect resveratrol production in hairy root culture of grape, and this strategy could be used to increase low resveratrol production in grapes.     

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)     

Artemisinin production in Artemisia annua hairy root cultures with improved growth by altering the nitrogen source in the medium

Murashige & Skoog medium was modified for enhancing artemisinin production in Artemisia annua hairy root cultures by altering the ratio of NO ₃ ^– /NH ₄ ⁺ and the total amount of initial nitrogen. Increasing ammonium to 60 mM decreased both growth and artemisinin accumulation in hairy root cultures. With NO ₃ ^– /NH ₄ ⁺ at 5:1 (w/w), the optimum concentration of total initial nitrogen for artemisinin production was 20 mM. After 24 days of cultivation with 16.7 mM nitrate and 3.3 mM ammonium, the maximum artemisinin production of hairy roots was about 14 mg l^–1, a 57% increase over that in the standard MS medium.