Horseradish Hairy Roots Research Articles

Variability of Bioactive Glucosinolates, Isothiocyanates and Enzyme Patterns in Horseradish Hairy Root Cultures Initiated from Different Organs.

Horseradish hairy root cultures are suitable plant tissue organs to study the glucosinolate–myrosinase–isothiocyanate system and also to produce the biologically active isothiocyanates and horseradish peroxidase, widely used in molecular biology. Fifty hairy root clones were isolated after Agrobacterium rhizogenes infection of surface sterilized Armoracia rusticana petioles and leaf blades, from which 21 were viable after antibiotic treatment. Biomass properties (e.g., dry weight %, daily growth index), glucosinolate content (analyzed by liquid chromatography-electronspray ionization-mass spectrometry (LC-ESI-MS/MS)), isothiocyanate and nitrile content (analyzed by gas chromatography-mass spectrometry (GC-MS)), myrosinase (on-gel detection) and horseradish peroxidase enzyme patterns (on-gel detection and spectrophotometry), and morphological features were examined with multi-variable statistical analysis. In addition to the several positive and negative correlations, the most outstanding phenomenon was many parameters of the hairy root clones showed dependence on the organ of origin. Among others, the daily growth index, sinigrin, glucobrassicin, 3-phenylpropionitrile, indole-3-acetonitrile and horseradish peroxidase values showed significantly higher levels in horseradish hairy root cultures initiated from leaf blades.

Strong antioxidant capacity of horseradish hairy root cultures under arsenic stress indicates the possible use of Armoracia rusticana plants for phytoremediation

The potential contamination of the food chain is the most important aspect of arsenic (As) pollution, since it is highly toxic to all organisms. Thus, the search for As hyperaccumulators suitable to remove As from contaminated soils appears to be a vital task. Horseradish (Armoracia rusticana), a crop plant with a high potential to accumulate heavy metals, can also serve to study the physiological processes that accompany arsenic stress. The significant adverse effect caused by arsenic exposure is an oxidative stress. Plants have developed a highly organized system to quench free radicals, which includes the action of both enzymatic and non-enzymatic quenching. Saccharides are proposed to possess outstanding antioxidant activity in vitro, and thus, they are likely to effectively quench free radicals also in plant tissues. In this study, root cultures (hairy root type) of horseradish were grown in vitro on media with different concentrations of arsenic (5–60 µg l−1). Arsenic slowed down the growth, nevertheless up to three-fold biomass increase was achieved at the highest dose. Moreover, root tissues were able to remove as much as 75% of arsenic from the cultivation medium within 7 days. We also evaluated diverse oxidative-stress-related features: contents of reactive oxygen species, the activities of key antioxidant enzymes, and the contents of important antioxidant molecules, such as glutathione, proline, phenolic compounds and non-structural carbohydrates. At all arsenic treatments, we observed a significant proline content increase and enhanced antioxidant enzymes (peroxidase, catalase and glutathione-S-transpherase) activities peaking, however, at different doses. Soluble carbohydrates contents also significantly increased after 7-day treatment a then decreased nearly to the original levels. This study points to efficient antioxidant system of horseradish hairy roots enabling good growth and substantial As accumulation even under high As exposure. Providing that horseradish shares these important features with this model system, we could propose that horseradish is a promising candidate to exploit in arsenic phytoremediation.

Metabolism of carbamazepine in plant roots and endophytic rhizobacteria isolated from Phragmites australis

Carbamazepine (CBZ) is a pharmaceutical frequently categorized as a recalcitrant pollutant in the aquatic environment. Endophytic bacteria previously isolated from reed plants have shown the ability to promote growth of their host and to contribute to CBZ metabolism.In this work, a horseradish (Armoracia rusticana) hairy root (HR) culture has been used as a plant model to study the interactions between roots and endophytic bacteria in response to CBZ exposure. HRs could remove up to 5% of the initial CBZ concentration when they were grown in spiked Murashige and Skoog (MS) medium. Higher removal rates were observed when HRs were inoculated with the endophytic bacteria Rhizobium radiobacter (21%) and Diaphorobacter nitroreducens (10%). Transformation products resulting from CBZ degradation were identified using liquid chromatography–ultra high-resolution quadrupole time of flight mass spectrometry (LC-UHR-QTOF-MS). CBZ metabolism could be divided in four pathways. Metabolites involving GSH conjugation and 2,3-dihydroxylation, as well as acridine related compounds are described in plants for the first time.This study presents strong evidence that xenobiotic metabolism and degradation pathways in plants can be modulated by the interaction with their endophytic community. Hence it points to plausible applications for the elimination of recalcitrant compounds such as CBZ from wastewater in CWs.

Clone selection of in vitro horseradish hairy root cultures

Clone selection of in vitro horseradish hairy root cultures

Identification of Plant Metabolites of Environmental Contaminants by UPLC-QToF-MS: The in Vitro Metabolism of Triclosan in Horseradish

Plants can extensively transform contaminants after uptake through phase I and phase II metabolism to a large diversity of products. UPLC-QToF-MS was used to detect and identify metabolites of the bacteriostatic agent triclosan in a horseradish hairy root culture. Thirty-three metabolites of triclosan were recognized by a stepwise approach of mass defect filtering, multivariate data analysis, and isotope pattern filtering from a data set of several thousands of signals in the exposed culture. Structure proposals were elaborated for 23 triclosan metabolites on the basis of their MS data. The majority were identified as conjugates (phase II metabolites) such as saccharides or sulfosaccharides. Additionally, a disulfosaccharide was identified as a plant metabolite for the first time. Besides that, also conjugates of a phase I metabolite, hydroxytriclosan, were determined in horseradish tissue extracts. Dehalogenation products of triclosan were not observed. The large number of metabolites detected and identified in this study emphasizes the importance of a comprehensive analytical approach in studies on the uptake and fate of organic contaminants in plants.

Effects of Sugar and Osmotic Pressure on Differentiation of Adventitious Shoots in Horseradish Hairy Roots

Horseradish (Armoracia rusticana) hairy roots were established by transformation with the Ri plasmid T-DNA in Agrobacterium rhizogenes 15834. To elucidate the effects of sugar and osmotic pressure on the differentiation of adventitious shoots in horseradish hairy roots, the hairy-root clone AR450 was cultured in phytohormone-free LS medium containing sucrose, glucose,and fructose under light conditions. Adventitious shoots were formed in the presence of 1.5% (W/V) glucose or 1.5% (W/V) fructose as well as in the presence of 3% (W/V) sucrose. High osmotic pressure inhibited the formation of adventitious shoots in horseradish hairy roots. Adventitious shoots were also formed effectively in liquid LS medium without phytohormones under light conditions.

Effects of heavy metals and nitroaromatic compounds on horseradish glutathione S-transferase and peroxidase

Glutathione S-transferase (GST) and peroxidase (POX) activities have a direct relation to the effect of stress on plant metabolism. Changes in the activities of the enzymes were therefore studied. Horseradish hairy roots were treated by selected bivalent ions of heavy metals (HMs) and nitroaromatic compounds (NACs). We have shown differences in GST activity when assayed with substrates 1-chloro-2,4-dinitrobenzene (CDNB) and 1,2-dichloro-4-nitrobenzene (DCNB). The conjugation of DCNB catalysed by GST was inhibited in all roots treated with HMs as compared to non-treated roots, whereas NACs caused induction of the activity in dependence on the exposition time and concentration of compounds. The conjugation of CDNB by GST was not affected to the same extent. The increase of GST activity was determined in cultures treated by nickel (0.1 mM) and diaminonitrotoluenes (DANTs, 0.1 mM) for 6 h, whereas the roots treated by 2,4,6-trinitrotoluene (TNT), 4-amino-2,6-dinitrotoluene (ADNT) and dinitrotoluene (DNT, 1.0 mM) needed 27 h treatment to induce the activity. The POX activity of cultures treated by HMs was inhibited to 17–35% in comparison to non-treated cultures. The POX activity of roots treated by TNT (0.1 and 1.0 mM) for 6 and 27 h and by ADNT (0.1 and 1.0 mM) for 6 h was inhibited. A partial increase of POX activity was measured in roots treated by all NACs for 27 h. The content of oxidized glutathione (GSSG) and reduced glutathione (GSH) in the roots differed significantly. It was followed as a symptom of the stress reaction of the plant metabolism to the effect of NACs and HMs.

Reduction of phytochrome level and light-induced formation of adventitious shoots by introduction of antisense genes for phytochrome A in horseradish hairy roots

In 5′; non-coding regions of genes for phytochrome A from horseradish (ArPHYAs) were fused with the 35S promoter of cauliflower mosaic virus in the antisense direction (CaMV35SantiArPHYAs) and introduced into horseradish hairy roots. Phytochrome levels of proximal areas in many hairy roots that had been transformed with CaMV35SantiArPHYAs decreased to levels of about one-half to one-quarter those of control hairy roots. The extent of the light-induced formation of adventitious shoots from hairy roots with less than half of the control level of phytochrome was lower than in the controls and not different between the three ArPHYAs. In contrast, the efficiency of phytochrome on the extent of shoot formation differed in hairy roots transformed with CaMV35SantiArPHYAs when phytochrome levels were more than 0.02 (Δ;(Δ;A) g−1). The efficiency of ArPHYA3 to initiate shoot formation was greatest and that of ArPHYA2 was smallest. Furthermore, previous reports on hairy roots overexpressing ArPHYAs showed a similar efficiency of phytochrome on shoot formation. These results indicate that the ArphyA1 and/or ArphyA3 play major roles in the light-induced formation of adventitious shoots and that ArphyA2 has a minor role.

Phytochrome Activities and Biological Activities of Genes for Phytochrome A of Horseradish in Horseradish Hairy Roots.

Full-length cDNAs of ArPHYAs were fused with the 35S promoter of cauliflower mosaic virus (CaMV35SArPHYAs) and introduced into horseradish hairy roots. The phytochrome levels in hairy roots that had been transformed with CaMV35SArPHYAs were about 2-3 times higher than those in normal hairy roots. Though ArphyAs were photochemically active in horseradish hairy roots, the efficiency of ArphyAs on shoot formation was different among them. The efficiency of light-induced formation of adventitious shoots in horseradish hairy roots was greatest for ArphyA3 and smallest for ArphyA2. These results indicated that ArPHYAs isolated from horseradish encoded photochemically active phytochromes and that they were biologically active in horseradish.

Segmental distribution in potentials of lateral root budding and oxygen uptake of plant hairy roots

The positional distributions in potential of lateral root budding and oxygen uptake rate were examined using the segments of madder and horseradish hairy roots with a length of 5.0×10 −3 m obtained at different mean distances from the root tips of l=7.5×10 −3–47.5×10 −3 m. The average rate of lateral root budding and oxygen uptake rate of the roots with smaller l values were higher and both the rates gradually decreased with increase in l value. Positive relations were observed between the rates of lateral root budding and oxygen uptake of both the hairy roots. The relation indicated that the potential of lateral root budding was suppressed at the oxygen uptake rates of 0.15×10 −5 and 0.32×10 −5 mol O 2/(h m) for madder and horseradish hairy roots, respectively.

Quantitative correlation between the concentration of photoreactive phytochrome and light-induced formation of adventitious shoots in horse-radish hairy roots

The hairy roots of horse-radish (Armoracia rusticana Gaert., Mey. et Scherb.) were found to contain spectrophotometrically active phytochrome. The absorption maxima of the red-absorbing and the far red-absorbing forms of phytochrome were the same as those of phytochrome I in etiolated tissues and of purified phytochrome A. Red light dramatically decreased the concentration of phytochrome in the hairy roots. The phytochrome was concentrated at the proximal ends of hairy roots and the concentration of phytochrome at each position in hairy roots was quantitatively correlated with the frequency of the light-induced formation of adventitious shoots at the same position. The level of phytochrome reflected the duration of culture in darkness. The increase in the concentration of phytochrome with time in darkness was positively correlated with the frequency of light-induced formation of adventitious shoots. The involvement of phytochromes A and B in the induction by light of budding of adventitious shoots from horse-radish hairy roots is discussed on the basis of the newly revealed correlations and earlier results.

Quantitative correlation between the concentration of photoreactive phytochrome and light-induced formation of adventitious shoots in horse-radish hairy roots

Quantitative correlation between the concentration of photoreactive phytochrome and light-induced formation of adventitious shoots in horse-radish hairy roots

Overexpression of phytochrome A enhances the light-induced formation of adventitious shoots on horseradish hairy roots

A full-length cDNA of the gene for phytochrome A from Arabidopsis thaliana was fused with the 35S promoter of cauliflower mosaic virus (CaMV35S-PHYA) and introduced into horseradish (Armoracia rusticana Gaert., Mey. et Scherb.) hairy roots. The phytochrome level in hairy roots transformed with CaMV35SPHYA was about three times greater than that in normal hairy roots and the rate of light-induced formation of adventitious shoots was also higher in the hairy roots transformed with CaMV35SPHYA. These results indicate that the light-induced formation of adventitious shoots on horseradish hairy roots is closely related to the phytochrome level.

Response to cytokinin in light-induced formation of adventitious shoots from horseradish hairy roots.

Response to cytokinin in light-induced formation of adventitious shoots from horseradish hairy roots.

24 Response to cytokinin in light-induced formation of adventitious shoots from horseradish hairy roots

24 Response to cytokinin in light-induced formation of adventitious shoots from horseradish hairy roots

Cryopreservation of horseradish hairy root cultures by encapsulation-dehydration

Horseradish hairy root cultures were successfully cryopreserved by encapsulation-dehydration. Root tips excised from the cultures were encapsulated in Ca-alginate beads containing 0.5 M glycerol and 0.3 M sucrose, and then the beads were slowly dehydrated by air-drying over silica gel to reduce the water content to 33% prior to cooling in liquid nitrogen. Preculture and dehydration of the root tips in the presence of 2 μM abscisic acid was effective in increasing the survival rate, regeneration of hairy roots being observed in about 60% of the tips dehydrated and cooled in liquid nitrogen.

Performance of a shaking vessel-type bioreactor with a current pole

The performance of a shaking vessel-type bioreactor with a current pole at its central axis was examined experimentally with respect to particle dispersion, power consumption, and solid-liquid mass transfer coefficient. The current pole improved the particle dispersion without an increase in power consumption and reduced the critical circulating frequency for complete suspension. The mass transfer coefficient with a current pole had the same value as that without a current pole above the critical circulating frequency for complete suspension. The shaking vessel-type bioreactor with a current pole was thus very suitable for the mass production of regenerated plantlets from fragmented horseradish hairy roots.

Conservation of Root Regeneration Potential of Cell Aggregates from Horseradish Hairy Roots Used as Artificial Seeds.

The effects of water content in agar gel used as a medium and oxygen level in the gas phase on the adventitious root regeneration of cell aggregates (CA) derived from horseradish hairy roots were investigated in cultures at 25° C. The number of roots emerging from CA was highly dependent on water content of the agar gel and no root regeneration was observed at a gel water content of 66% during culture time of 20 days. CA root regeneration was suppressed when the CA were kept for 20 days in an atmosphere oxygen composition of 10%, but was restored upon transfer of the CA to normal atmosphere of 21% oxygen. On the basis of these findings, an artificial seed was proposed using the CA as a cell inclusion material encapsulated in alginate gels covered with coats. From the perspective of conserving the root regeneration potential of the CA by preventing the drying of alginate gel while keeping oxygen availability to the CA, different coating materials of ethylene vinyl acetate acrylic acid terpolymer, paraffin and polyorganosiloxane were tested. Paraffin was selected as a suitable coating material because of its efficient drying tolerance and adequate permeability to oxygen. A regeneration efficiency of 90% could be obtained from the CA, stored in alginate gel covered with a paraffin coating of 0.40 mm thickness at 25° C for 60 days in air, when sucrose concentration in the gel was over 240 mol m -3 .

Efficient culture method for production of plantlets from mechanically cut horseradish hairy roots

Effects of the initial density of roots fragmented by blender cutting and the addition of kinetin on plantlet formation were studied. The number of plantlets increased with an increasing initial density of root fragments (1.7 g-f.w./ l to 13.3 g-f.w./ l) at 0.1 mg/ l of kinetin, but the number of plantlets with elongated roots unavailable for use as artificial seed dramatically increased after kinetin depletion in the medium. The condition at 0.8 mg/ l of kinetin in the medium and 13.3 g-f.w./ l of initial density of root fragments increased the number of the plantlets without roots (suitable for artificial seed) compared with the condition at 0.1 mg/ l of kinetin in the medium and 13.3 g-f.w./ l of initial density of roots. To avoid the toxic effect of a high concentration of kinetin at the beginning stage of the culture, 0.1 mg/ l of kinetin was periodically supplemented in the medium 8 times (on the initial day and from 4 d to 10 d of the culture). Under this condition, the total number of plantlets increased and reached a maximum on 12 d, and the number of plantlets 2–4 mm in size without roots (6,270 l −1) was the highest of all other cultures.

Effects of light and growth regulators on adventitious bud formation in horseradish (Armoracia rusticana)

To clarify that the presence of Ri T-DNA genes are not prerequisite for the light-induced bud formation in horseradish (Armoracia rusticana) hairy roots, leaf and root segments of nontransformed horseradish plants were used as explants. Bud formation from nontransformed tissues was observed in hormone-free medium under 16 h daylight conditions, but not under continuous darkness. To investigate the effects of growth regulators on bud formation, leaf and root explants were treated with auxin (1-naphthaleneacetic acid; NAA) and / or cytokinin (6-benzyl-aminopurine; BA). The most effective treatment in the dark to stimulate bud formation was BA at 1 mg·1(-1). These results show that adventitious bud formation in horseradish can be induced by light and growth regulators, and especially cytokinin, may be involved in bud formation, irrespective of whether the tissues were transformed with Ri T-DNA.