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     

Optimized genetic transformation of Zanthoxylum zanthoxyloides by Agrobacterium rhizogenes and the production of chelerythrine and skimmiamine in hairy root cultures

Zanthoxylum zanthoxyloides is an endangered African tree producing numerous bioactive substances including antileukemic and antisickling agents. Here, the potential of Z. zanthoxyloides hairy root cultures was tested for the production of bioactive substances with limited natural resources. The efficiency of Agrobacterium rhizogenes LBA9402‐mediated transformation of leaf material was evaluated using different techniques. An optimal transformation frequency of 77% was obtained after 11 days by inoculating A. rhizogenes directly onto the central vein of 14‐week‐old leaves followed by a co‐cultivation period of 3 days. Different treatments in immersion mode (manual wounding, acetosyringone, CaCl₂, ultrasonication) never exceeded these results. A maximum growth rate of 0.37 cm/day was determined during the exponential phase. Liquid chromatography‐diode array detection analysis showed the presence of skimmiamine, sesamine, chelerythrine, and chelerythrine derivatives in Z. zanthoxyloides hairy root lines. The maximum production of skimmiamine and chelerythrine in 28‐day‐old hairy root cultures was 45 ± 2 and 107 ± 4 mg/100 g dry weight, respectively. The present results highlight the potential of Z. zanthoxyloides hairy root cultures for the sustainable production of skimmiamine and chelerythrine.     

Enhanced production of flavonolignans in hairy root cultures of Silybum marianum by over-expression of chalcone synthase gene

In the present study, metabolic engineering approach was used through over-expressing the Petunia chalcone synthase (chsA) gene in order to enhance the silymarin production level in the hairy root cultures of Silybum marianum. Molecular analysis confirmed the presence and integration of chsA transgene in transgenic hairy roots. Chemical analysis indicated that the over-expression of chsA gene enhanced the silymarin production level in the transgenic line as much as 7-folds than the non-transgenic hairy roots. Moreover, the silybin content, the main active component of silymarin, was proved to be 10 times higher in transgenic hairy roots than those of the non-transgenic ones. Therefore, the over-expression of petunia chsA gene in S. marianum hairy roots did not result in gene silencing, but led to an enhanced biosynthesis of the flavonolignans.     

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

Before the late 1980s, although the majority of Agrobacterium-mediated gene transfer experiments have been performed with A. tumefaciens[1―3], some work has also been done with its close relative, Agro-bacterium rhizogene. It has been considered that onl…     

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

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

Indole alkaloid production by hairy root cultures of Catharanthus roseus

Hairy root cultures of Catharanthus roseus were established by infection with six different Agrobacterium rhizogenes strains. Two plant varieties were used and found to exhibit significantly different responses to infection. Forty-seven hairy root clones derived from normal plants and two derived from the flowerless variety were screened for their growth and indole alkaloid production. The growth rate and morphological appearance showed wide variations between the clones. The alkaloid spectra observed were qualitatively but not quantitatively very similar to that of the corresponding normal plant roots. No vindoline or deacetyltransferase activity could be detected in any of the cultures studied. O-acetylval-lesamine, an alkaloid which has not been previously observed in C. roseus was identified from extracts of hairy root clone No. 8. Two root clones were examined for their growth and alkaloid accumulation during a 26-day culture period. Alkaloid accumulation parallelled growth in both clones with ca. 2 mg ajmalicine and catharanthine per g dry weight being observed.Dedicated to Dr. Friedrich Constabel on the occasion of his 60th birthday     

Cytogenetic analysis of hairy root cultures from a number of plant species transformed by Agrobacterium rhizogenes

Hairy root cultures, obtained after transformation of seven plant species by A. rhizogenes, were examined cytologically to assess their chromosome number. All species had the correct 2n diploid number of chromosomes in root tip cells. Free cell suspensions of two of the species were also examined and were found to be variable with polyploids or aneuploids predominating. The DNA from the hairy root cultures was isolated and the presence of the Agrobacterium T-DNA confirmed by Southern blotting. The relevance of these observations to the use of hairy roots in the study of plant secondary metabolite production is discussed.     

Perturbation of alkaloid production by cadaverine in hairy root cultures of Nicotiana rustica

Cadaverine (1–10 mM) stimulated the production of anabasine by hairy root cultures of Nicotiana rustica transformed with Agrobacterium rhizogenes. In control cultures, nicotine accounted for at least 70–80% of the total alkaloid produced, whereas in cultures supplemented with 5 mM cadaverine about two-thirds of the alkaloid was anabasine and nicotine production was markedly diminished. Putrescine and agmatine caused some stimulation of alkaloid production, but the ratio of nicotine to anabasine was essentially unaffected. Lysine caused no substantial increase in anabasine formation.     

Enhanced production of isoflavones by elicitation in hairy root cultures of Soybean

A combined treatment of sonication (2 min) and vacuum infiltration (2 min) stimulated isoflavones production of 75.26 mg g^?1 DW which was 15.11-fold higher than control hairy root line at optimal harvest time of 40 days. Addition of MeJ at 100 μM concentration with 72 h exposure time on 30 day-old hairy root culture further enhanced total isoflavones production of 53.16 mg g^?1 DW (10.67-fold) and SA at 200 μM concentration with 96 h exposure period enhanced the production of isoflavones (28.79 mg g^?1 DW; 5.78-fold). MeJ-treated hairy roots reduced biomass accumulation whereas sonication, vacuum infiltration and SA did not exhibit a negative effect on biomass growth.     

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.     

Tropane alkaloid production by hairy roots of Atropa belladonna obtained after transformation with Agrobacterium rhizogenes 15834 and Agrobacterium tumefaciens containing rol A, B, C genes only

Atropa belladonna leaf disks were infected by a wild strain Agrobacterium rhizogenes 15834 harboring the Ri-TL-DNA and by a disarmed Agrobacterium tumefaciens strain harboring a construction with only rol ABC and npt II genes. Thirteen root lines were established and examined for their growth rate and alkaloid productivity to evaluate the possible role of rol genes in morphological differentiation and in tropane alkaloid formation. A great diversity has been observed in the growth rate of these 13 root lines. The root biomass increased up to 75 times. The total alkaloid contents were similar in the root lines obtained by infection with A. rhizogenes 15834 and A. tumefaciens rol ABC. The last ones accumulated between 4 (1.1 mg g(-1) DW) and 27 (8 mg g(-1) DW) times more alkaloids than the intact roots (0.3 mg g(-1) DW). This work has shown that the rol ABC genes were sufficient to increase tropane alkaloid production in A. belladonna hairy root cultures.     

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     

Indole alkaloid production by hairy root cultures ofCatharanthus roseus: Growth kinetics and fermentation

Summary Growth kinetics and indole alkaloid production ofCatharanthus roseus hairy root cultures were studied in shake flasks and in a small scale fermenter. A logistic growth model commonly used for microbes described well the growth of hairy roots. Of the several parameters analyzed during the cultivation of hairy roots, a linear relationship between sucrose consumption and dry weight increase was obtained. This suggests the validity of sugar analysis as a means in monitoring the growth of hairy roots in fermenters.     

Efficiency of Agrobacterium rhizogenes–mediated root transformation of Parasponia and Trema is temperature dependent

Parasponia trees are the only non-legume species that form nitrogen-fixing root nodules with rhizobium. Based on its taxonomic position in relation to legumes (Fabaceae), it is most likely that both lineages have gained this symbiotic capacity independently. Therefore, Parasponia forms a bridging species to understand the evolutionary constraints underlying this symbiosis. However, absence of key technologies to genetically modify Parasponia seriously impeded studies on these species. We employed Agrobacterium rhizogenes to create composite Parasponia andersonii plants that harbour transgenic roots. Here, we provide an optimized protocol to infect P. andersonii as well as its non-symbiotic sister species Trema tomentosa with A. rhizogenes. We show that the transformation efficiency is temperature dependent. Whereas the optimal growth temperature for both these species is 28?°C, the transformation is most efficient when co-cultivation with A. rhizogenes occurs at 21?°C. Using this optimized protocol up to 80?% transformation efficiency can be obtained. These robust transformation platforms will provide a strong tool to unravel the Parasponia–rhizobium symbiosis.     

Saponin production by cultures of Panax ginseng transformed with Agrobacterium rhizogenes

Hairy root culture of Ginseng (Panax ginseng) was established after roots were induced on callus following infection with Agrobacterium rhizogenes. The transformed cultures of ginseng could be subcultured as an axenic root culture in the absence of phytohormones, and grew with extensive lateral branches more rapidly than the ordinary cultured roots induced by hormonal control from ginseng callus. The hairy roots synthesized the same saponins, ginsenosides, as those of the native root, up to about 2.4 times in the quantity, and up to about 2 times in comparison with that of the ordinary cultured roots, on dry weight basis.Abbreviations Ms medium Murashige and Skoog's medium - 2,4-D 2,4-dichloro-phenoxyacetic acid - IBA indole-3-butyric acid - K kinetin This paper is Part 52 in the series "Studies on Plant Tissue Culture". For Part 51, see Ayabe S, Udagawa A, Furuya T (1987).     

Identification of hairy root loci in the T-regions of Agrobacterium rhizogenes Ri plasmids

Summary Agrobacterium rhizogenes induces root formation at the wound site of inoculation in plants and inserts a fragment of its plasmid (Ri) into the plant nuclear DNA. Parts of the transferred region (T-region) of the Ri plasmid of A. rhizogenes strain A4 or 8196 are cloned in Escherichia coli. Insertions of the E. coli lacZ coding region into the hybrid plasmids were made in vivo using transduction by miniMu. Twenty insertions localized in the T_L-DNA of pRiA4 (or pRi1855) and 2 inserts in the T-DNA of pRi8196 were obtained in E. coli. One of the T_L-DNA insertions is saved up because it is linked to an internal T-DNA deletion; the others because they confer a lactose plus phenotype on E. coli; this indicates that the T-DNA harbours sequences that are expressed in E. coli. Fifteen of these T-DNA insertions were transfered to Agrobacterium where they substitute the corresponding wild-type T-DNA of the Ri plasmid by homologous recombination. These strains corresponding to insertion-directed mutagenesis were used to inoculate Daucus carota slices and stems and leaves of Kalanchoe daigremontiana. The two insertions strains obtained in the T-DNA of pRi8196 are avirulent on K. daigremontiana; but their phenotypes differ on D. carota slices, suggesting that insertions affect distinct loci on the T-DNA involved in hairy root formation. Only one insertion out of the twenty obtained in the T_L-DNA of pRiA4 (or 1855) induces a loss of virulence on leaves of K. daigremontiana. However the T_L-DNA deletion harbouring strain induces a loss of virulence on D. carota and K. daigremontiana (stems and leaves), confirming the importance of the T_L-DNA for hairy root induction. re]19850711 rv]19851230 ac]19860114