Hairy Root Lines Research Articles

Increasing tryptophan synthesis in a forage legume Astragalus sinicus by expressing the tobacco feedback-insensitive anthranilate synthase (ASA2) gene.

A cDNA clone that encodes a feedback-insensitive anthranilate synthase (AS), ASA2, isolated from a 5-methyl-tryptophan (Trp) (5MT)-resistant tobacco cell line under the control of the constitutive cauliflower mosaic virus 35S promoter, was introduced into the forage legume Astragalus sinicus by Agrobacterium rhizogenes with kanamycin selection. The 35S-ASA2 gene was expressed constitutively as demonstrated by northern-blot hybridization analyses and the presence of feedback-insensitive AS. Hairy root lines transformed with 35S-ASA2 grew in concentrations of up to 100 microM 5MT, whereas the controls were completely inhibited by 15 microM 5MT. Expression of the feedback-insensitive ASA2 resulted in a 1.3- to 5.5-fold increase in free Trp. Kinetic studies of the AS activity demonstrate the Trp feedback alterations and indicate that the ASA2 alpha-subunit can interact with the native A. sinicus beta-subunit to form an active enzyme. The ASA2 transcript and high free Trp were also detected in the leaves, stems, and roots of plants regenerated from the transformed hairy roots. Thus, we show for the first time that ASA2 can be used to transform plants of a different species to increase the levels of the essential amino acid Trp and impart 5MT resistance.

Production of transgenic plants via Agrobacterium rhizogenes-mediated transformation of Panax ginseng.

Hairy roots of Panax ginseng were obtained after root disks were infected with wild-type strain Agrobacterium rhizogenes 15834. Three lines of hairy roots with different pigmentation were selected. Embryogenic callus was induced on Murashige and Skoog medium containing 1.0 mg/l 2,4-D. The frequency of embryogenic callus formation was 37.4% in a line with red pigmentation. Somatic embryo development from embryogenic callus was efficiently achieved by lowering the concentration of 2,4-D (0.5 mg/l). After the germination of somatic embryos on medium with 10 mg/l GA3, the embryos were transferred to 1/2-MS medium without GA3. The transformed ginseng plantlets had an actively growing root system with abundant lateral roots. The phenotypical alteration of transformed ginseng plants might be valuable character for increasing root yield.

A hairy root culture of melon produces aroma compounds.

Musk melon is the favorite fruit with a high market value in Japan, and the fragrance is one of the major factors determining the fruit quality of melon. In this study, mutant melon hairy roots which had been induced by means of the T-DNA insertion mutagenesis were found to produce volatile compounds with the fruity fragrance of mature melon. The volatile compounds were extracted and identified by GLC-mass spectrometry. Some essential oils such as (Z)-3-hexenol, (E)-2-hexenal, 1-nonanol, and (Z)-6-nonenol were stably synthesized by these hairy roots despite the increased number of subcultures. The productivity of these compounds by the best hairy root line was shown to be considerably higher than naturally ripened melon fruits.

Hairy root production in Arabidopsis thaliana: cotransformation with a promoter-trap vector results in complex T-DNA integration patterns.

In comparison with the production of transgenic plants, the generation of hairy roots has the advantage that more independent transgenic lines can be produced in a shorter period of time. Therefore, we wanted to combine this approach with the promoter-trapping strategy to identify nematode-induced plant promoters. For the efficient production and culture of transgenic hairy root lines of Arabidopsis thaliana, the standard Agrobacterium rhizogenes transformation procedure was modified to avoid rapid callusing of the hairy roots. An average of 0.72 independent kanamycin-resistant (KmR) roots were obtained per leaf piece. However, a much lower frequency of reporter gene activation was obtained than expected from experiments with the same vectors in Agrobacterium tumefaciens: of more than 700 independent KmR hairy roots tested, only 8 were β-glucuronidase (GUS) positive. DNA hybridization was done on ten hairy root lines, of which one had a single truncated T-DNA and the others multiple copies of T-DNA that led to complex hybridization patterns. In a parallel analysis of A. thaliana plants transformed with the same vectors using A. tumefaciens, relatively simple T-DNA integration patterns were obtained. The low occurrence of GUS-positive hairy root lines in our experiments could be explained by the multiple T-DNA copies, especially in inverted array, that result in high frequencies of gene inactivation.

Datura metel: in vitro production of tropane alkaloids.

Hairy root lines of Datura metel were established following infection of aseptic stem segments with Agrobacterium rhizogenes strain A4 and cultured in hormone-free B5 solid medium. The growth and production of hyoscyamine and scopolamine (mg/g dry wt.) of these root cultures was encouraged by using B5 liquid medium with half-strength salts. In these culture conditions, the capacity of the highest productive root line 25 to excrete scopolamine into the culture medium rose from 8.7% to 70% when the permeabilizing agent Tween 20 was added for 24 h to the medium, after 2 and 4 weeks of culture. Using an airlift bioreactor (41) with modifications in order to increase root anchorage, the Tween 20 treatment encouraged both growth and alkaloid productivity of cultured root line 25. After 4 weeks their biomass yield was 2.3 mg/l/day and 0.84 mg/l/day of scopolamine was produced (70% extracellular). The scopolamine released into the culture medium was separated with an Amberlite XAD-2 column located in the media exit.

Influence of calcium ion-concentration in the medium on tropane alkaloid accumulation in Datura stramonium hairy roots

Hairy root cultures of Datura stramonium were established after the infection of stem sections with Agrobacterium rhizogenes A4, and cultured in phytohormone free B 5 liquid medium containing different calcium concentrations in order to investigate the relationship between Ca 2+-dependent peroxidase activity and hyoscyamine accumulation. Putrescine:SAM N-methyltransferase (PMT) activity and the levels of its mRNA were determined. Although calcium concentrations below that present in B 5 medium (1.0 mM) did not affect hairy root line growth, they significantly reduced root peroxidase activity which is probably involved in the degradation of secondary products. Lower calcium concentrations also reduced the hyoscyamine synthesis ability of the hairy root lines. This loss of hyoscyamine production was concurrent with the loss of PMT activity, which is supported by Northern blot analysis performed on the total RNA fraction using cDNA corresponding to PMT of tobacco as probe.

Influence of Agrobacterium rhizogenes Strains on Biomass and Alkaloid Productivity in Hairy Root Lines of Hyoscyamus muticus and H. albus *

Using leaf explants of IN VITRO grown HYOSCYAMUS ALBUS and H. MUTICUS plantlets, hairy roots were induced following inoculation with AGROBACTERIUM RHIZOGENES strains A (4) and LBA-9402. The transformed roots, appearing after 14 - 17 days incubation on hormone-free MS medium containing 1 g/L cephalexin, were excised and maintained in the same medium. Ten randomly selected hairy root lines from each bacterial treatment of the two plant systems were compared for growth and alkaloid production in half-strength, hormone-free MS medium on 25 (th) day of culture. A. RHIZOGENES strain - A (4) induced hairy root lines of both H. ALBUS and H. MUTICUS were comparatively faster growing than those induced by strain LBA-9402. In contrast to earlier reports, some of the hairy root lines of H. ALBUS induced by A. RHIZOGENES strain A (4) were as fast growing as the hairy root lines of H. MUTICUS. The atropine yields of A (4) induced lines of H. ALBUS were significantly higher (3.5 fold) than the LBA-9402 induced lines. No such relationship between the bacterial strain and alkaloid productivity could, however, be obtained in case of hairy root lines of H. MUTICUS.

Ri-mediated transformation of Artemisia annua with a recombinant farnesyl diphosphate synthase gene for artemisinin production

A transgenic system was developed for Artemisia annua L. via Agrobacterium rhizogenes-mediated transformation. Using this system a cDNA encoding farnesyl diphosphate synthase (FDS) placed under a CaMV 35S promoter was transferred into Artemisia annua using Agrobacterium rhizogenes strain ATCC15834. Among the 150 hairy root lines established, 16 lines showed resistance to kanamycin (20 mg l-1). The intergration of FDS gene was confirmed by PCR and Southern blot analysis, and analysis of Northern blot revealed that the foreign FDS gene was expressed at the transcriptional level in three hairy root lines (F-1, F-24 and F-26 root line). F-1, F-24 and F-26 root lines grew faster than the control hairy root line. However, on the MS medium growth of F-26 root line was abnormal in that callus frequently formed. Analysis of artemisinin demonstrated that about 2–3 mg g-1 DW of artemisinin were then detected in the three root lines, which is about 3–4 times higher than that in the control hairy roots.

Nutrient consumption and alkaloid accumulation in a hairy root line of Catharanthus roseus

A hairy root line from Catharanthus roseus was cultured in a 14 l bioreactor. Nitrate and phosphate uptakes were similar to the same root line cultured in a 250 ml Erlenmeyer flask. However, sucrose consumption rate was slower in roots cultured in the bioreactor. These results show that it is feasible to upgrade this hairy root line to bioreactor level and, although carbon utilization has to be improved, ajmalicine and catharanthine were still produced and were retained within the biomass tissue.

Production of flavonoids by Psoralea hairy root cultures

Eighteen transformed root cultures from 7 Psoralea plant species (Leguminosae) were established with the objective of producing daidzein and related flavonoids. All the 18 hairy root lines grew fast and had the same capacities for biomass production. Each of them produced daidzein as an intracellular secondary metabolite. The Lach5 hairy root line, obtained from P. lachnostachys, was a high producing line for daidzein and was further studied for biomass and flavonoid production. This root line showed exponential growth. Chitosan was used for elicitation purposes as well as for its permeabilizing effect. Little elicitation effect could be demonstrated and the metabolite release in the medium was weak (about 1%) and limited to the first 29 h after chitosan addition. Daidzein was demonstrated to be more concentrated in young parts (apexes) whereas coumestrol content was higher in older parts (brown tissues). Compared to callus cultures from the same plant species, hairy roots displayed comparable concentrations. However, high-producing lines were more frequently found with hairy roots (4 out of 18) than with callus cultures (4 out of 217)

Characterization of transcription of genes involved in hairy root induction on pRi1724 core-T-DNA in two Ajuga reptans hairy root lines

The detailed status of the transcription of genes on pRi1724 T-DNA in two independent hairy root lines of the plant Ajuga reptans, Ar-4 and Ar-24, which have several different characteristics, was obtained by Northern blotting and a reverse transcription-polymerase chain reaction (RT-PCR) analysis. In the Northern blotting analysis using DNA fragments corresponding to the putative open reading frames (ORFs) as probes, transcripts from putative ORFs 10 ( 1724rolA), 11 ( 1724rolB), 12 ( 1724rolC), 13a and 14, which are homologs to each ORF on pRiA4, were detected in both hairy root lines, whereas no transcripts derived from ORF 13 were detected. The transcription of ORF 13 was, however, detected in the RT-PCR analysis, suggesting a minor expression of ORF 13. All of the putative ORFs were transcribed with their expected directions, since DNA fragments were amplified when the antisense primers were employed in the reverse transcription. We also found that different transcripts with a reverse direction were present at the locations of 1724rolA, 1724rolC and ORF 13a, because the DNA fragments were amplified from the templates when their sense primers were used in the reverse transcription.

Variation in the growth and alkaloid production capability of the hairy roots of Hyoscyamus albus, H. muticus and their somatic hybrid

Hairy root cultures have been established by inoculating the somatic hybrid ( Hyoscyamus muticus + H. albus) as well as parent leaf explants with two strains of Agrobacterium rhizogenes (A4 and LBA 9402). The transformation frequency obtained with the A4 strain was found to be better than that of the LBA 9402 strain in all three test systems. Six lines each of H. albus and H. muticus, and four lines of the hybrid were studied for their growth and secondary metabolite production after 30 days of culture. Great variation was observed among the various lines induced by the two strains of bacteria. The maximum biomass had been noted in the somatic hybrid line 2D of A4 origin which is 1.26 and 1.55 times higher than that of the maximum biomass yielding lines 3M and 43A of H. muticus and H. albus respectively of the same bacterial origin. The somatic hybrid hairy root line 1D induced by A4 strain revealed maximum yield of atropine (9.73mg/culture) which was 9.18 times and 1.45 times higher than that of the highest atropine yielding hairy root line of H. muticus (35M) and H. albus (28A) respectively, of A4 origin. The scopolamine content of the same somatic hybrid line was found to be 1.13 times higher than that of the maximum scopolamine yielding H. albus line (28A).

Enhanced production of valepotriates by Agrobacterium rhizogenes induced hairy root cultures of Valeriana wallichii DC

Hairy roots of Valeriana wallichii DC were obtained following co-cultivation of detached leaf explants with Agrobacterium rhizogenes strains A 4 and LBA 9402. For V. wallichii the A 4 strain appeared to be better than the LBA 9402 strain in terms of both relative rate of hairy root formation and growth of the respective hairy root line. The optimal growth of both the hairy root lines occurred on half strength MS [1]salt medium with 40 gm/l sucrose. Valepotriate contents from the roots of 24 week old wild type field grown plants were compared with those of 12, 16 and 20 week old hairy root lines induced by the A 4 and LBA 9402 strains of A. rhizogenes. The homodidrovaltrate, didrovaltrate, IVHD and acevaltrate contents were quantified individually by HPLC. The valepotriates were localized only in the root tissues and were not detected in the culture medium. In terms of the production of total as well as individual valepotriates, the LBA 9402 induced hairy root line appeared to be a better performer than the A 4 induced one. The total valepotriate content was highest in the 20 week old LBA 9402 induced hairy root line (8.52% dw) followed by that of the A 4 induced line (5.10% dw) of the same age, which were 3.3 and 2 times higher, respectively than that of the control roots (2.58% dw). Rapid growth of the hairy roots of V. wallichii with in vitro valepotriates production potential may offer an attractive alternative to the exploitation of this endangered plant species.

Establishment of transformed root cultures of Paulownia tomentosa for verbascoside production

The formation of eleven hairy root lines of Paulownia tomentosa was induced by infection of stem segments with strains of Agrobacterium rhizogenes LBA 9402 and A4. The transformation frequency was dependent on the age of expiant. Hairy root cultures grew rapidly with the characteristic high degree of lateral branching in hormone-free Woody Plant liquid medium. Production of verbascoside in hairy roots under culture conditions was demonstrated. Depending on the hairy root line used, the level of verbascoside varied from 1.7 to 8% of dry weight. Enhancement of verbascoside production was attempted by using various basal media, like Murashige and Skoog (MS), Gamborg (B5) and Woody Plant (WP) as well as half-strength MS, B5 and WP media. Experiments with one of the most productive line (PT-3) showed that the maximum verbascoside yield was achieved in half-strength Gamborg's B5 liquid medium. Under these conditions the transformed roots had a verbascoside content amounting to 9.5% of dry weight (more than 1 g/L of culture). The amount was approximately doubled compared with the yield in the roots of 4.5-month-old-plants.

Production of daidzein by callus cultures of Psoralea species and comparison with plants

Callus cultures were established from five Psoralea species (Leguminosae) with the objective of producing daidzein (isoflavone). The biomass doubling times ranged from 7 to 16 days according to the species and a 48 weeks period was necessary to obtain lines with stable growth characteristics. All the 217 callus lines were analyzed for their daidzein content using HPLC. Our callus collection showed a large interspecific variation and the highest concentrations were recovered in P. obtusifolia callus lines (maximum of 0.9680% DW). Intraspecific variation was also important and allowed the recovery of high-producing lines (production exceeding 0.3000% DW) with four out of the five Psoralea species studied. The daidzein repartition was investigated in planta with P. cinerea in order to evaluate the potential of in vivo production. Mature fruits were the richest organs for daidzein concentration in P. cinerea and were used as indicators to evaluate the possible production with the other four plant species. In vitro concentrations were always much higher than in planta, and no correlation could be established between the calluses and plants for the five species. Our callus lines contained concentrations comparable to Psoralea hairy root lines. They can be considered as an interesting material to further study the production of daidzein.

Regeneration of transgenic vegetable brassicas (Brassica oleracea andB. campestris) via Ri-mediated transformation.

A procedure for the production of fertile transgenic brassicas via Ri-mediated transformation is reported in this paper. Transgenic hairy root lines were selected for 12 vegetable brassica cultivars and lines representing six varieties: broccoli, Brussels sprouts, cabbage, cauliflower, rapid-cycling (allBrassica oleracea) and Chinese cabbage (B. campestris). Leaf explants or petioles of intact cotyledons were co-cultivated withAgrobacterium strain A4T harbouring various binary vectors. The T-DNA region of all binary vectors contained a neomycin phosphotransferase II gene for kanamycin resistance, in addition to other genes. Hairy root lines grew prolifically on hormone-free medium containing kanamycin. Transgenic shoots were regenerated from all cultivars either spontaneously or after transfer of hairy roots to a hormone-containing medium. Southern analysis confirmed that the plants were transgenic. Plants from all brassica types were successfully transferred to greenhouse conditions. Plants were fertile and segregation analysis confirmed transmission of traits to progeny.

メロン毛状根の緑化誘導条件の検討

The potential traits of photosynthetic and photoautotrophic growth of hairy root derived from co-cultures of Cucumis melo L. with Agrobacterium rhizogenes were investigated. Thirty-two different lines of hairy roots induced from leaf explants of the plant were used to determine the conditions which favor the induction of photoautotrophy. Although all of the hairy root lines remained white under dark condition, some of them turned yellow green to pale green when incubated in MS medium containing low concentration of sucrose and supplemented with a buffer releasing CO2. One of the initially heterotrophic hairy root cultures, KMH-18, turned green rapidly, produced chlorophyl, and grew well depending on CO2 fixation under these conditions.

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.

Agrobacterium rhizogenes-mediated transformation of scented geranium

A method is described for producing genetically transformed plants from explants of three scentedPelargonium spp. Transgenic hairy root lines were developed fromPelargonium spp leaf explants and microcuttings after inoculation withAgrobacterium rhizogenes strains derived from the agropine A4 strain. Hairy root lines grew prolifically on growth regulator-free medium. Transgenic shoots were regenerated from hairy roots and the plants have been successfully transferred to soil. The phenotype of regenerated plants has been characterized as having abundant root development, more leaves and internodes than the controls, short internodes and highly branched roots and aerial parts. Southern blot analyses have confirmed the transgenic nature of these plants.

Establishment and characterization of photosynthetic hairy root cultures of Datura stramonium

Twelve different lines of Datura stramonium (normal and hairy) root cultures were subjected to conditions which induce photoautotrophy. Two of the hairy root lines responded to induction, showing clearly a diminished growth rate when compared to heterotrophic cultures, an increase in chlorophyll, a net O2 evolution, CO2 fixation, and de novo synthesis of the ribulose 1,5 biphosphate carboxylase enzyme. A time course of growth and tropane alkaloid levels in the tissue and medium, revealed a correlation between the development of the photosynthetic apparatus and the increase in scopolamine. Although normal cultures did not grow photosynthetically, they showed some greening response under the first step of the induction. The correlation between development of photosynthesis and increase in scopolamine synthesis were corroborated with normal root cultures. This experimental model is used for the basic study of the regulatory enzymes involved in the biosynthesis of tropane alkaloids, as well as for the study of their mechanisms of transport.