Antioxidant enzyme activities and isozyme pattern in hairy roots and regenerated tobacco plants

Hairy root disease is caused by infection of wounded higher plants with Agrobacterium rhizogenes. Transformation of tissues or plants with A. rhizogenes, as well as transformation with rol genes, in addition to hairy roots, may produce alterations in the plant secondary metabolism. H₂O₂ and other ROS are involved as signals in secondary metabolite production pathways and play a key role in plant defense reactions. In this work the effects of A. rhizogenes rol genes on nicotine content, antioxidant enzymes activity, H₂O₂ production, the pattern of peroxidase (POX) and superoxide dismutase (SOD) isozymes in hairy roots and regenerated Nicotiana tabacum plants were studied. The rise in SOD and POX activities in the transformed lines TRa and TRb and the resulting regenerated plants and a decreased level of H₂O₂ in them as compared with the untransformed lines indicates that rol gene expression decreases H₂O₂ level probably by increasing production of antioxidant enzymes. A decreased H₂O₂ content in TRc line, in spite of similarity of antioxidant enzyme activity as compared to normal roots, indicates that rol genes activate other mechanisms except SOD and POX enzymes for reducing H₂O₂.     

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.     

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.     

<Emphasis Type="Italic">Agrobacterium rhizogenes</Emphasis>: recent developments and promising applications

Agrobacterium rhizogenes is the etiological agent for hairy-root disease (also known as root-mat disease). This bacterium induces the neoplastic growth of plant cells that differentiate to form “hairy roots.” Morphologically, A. rhizogenes-induced hairy roots are very similar in structure to wild-type roots with a few notable exceptions: Root hairs are longer, more numerous, and root systems are more branched and exhibit an agravitropic phenotype. Hairy roots are induced by the incorporation of a bacterial-derived segment of DNA transferred (T-DNA) into the chromosome of the plant cell. The expression of genes encoded within the T-DNA promotes the development and production of roots at the site of infection on most dicotyledonous plants. A key characteristic of hairy roots is their ability to grow quickly in the absence of exogenous plant growth regulators. As a result, hairy roots are widely used as a transgenic tool for the production of metabolites and for the study of gene function in plants. Researchers have utilized this tool to study root development and root–biotic interactions, to overexpress proteins and secondary metabolites, to detoxify environmental pollutants, and to increase drought tolerance. In this review, we provide an up-to-date overview of the current knowledge of how A. rhizogenes induces root formation, on the new uses for A. rhizogenes in tissue culture and composite plant production (wild-type shoots with transgenic roots), and the recent development of a disarmed version of A. rhizogenes for stable transgenic plant production.     

Recovery of phenotypically normal transgenic plants of Brassica oleracea upon Agrobacterium rhizogenes-mediated co-transformation and selection of transformed hairy roots by GUS assay

In this paper we describe the production of transgenic broccoli and cauliflower with normal phenotype using an Agrobacterium rhizogenes-mediated transformation system with efficient selection for transgenic hairy-roots. Hypocotyls were inoculated with Agrobacterium strain A4T harbouring the bacterial plasmid pRiA4 and a binary vector pMaspro::GUS whose T-DNA region carried the gus reporter gene. pRiA4 transfers T_L sequences carrying the rol genes that induce hairy root formation. Transgenic hairy-root production was increased in a difficult-to-transform cultivar by inclusion of 2,4-D in the medium used to resuspend the Agrobacterium prior to inoculation. Transgenic hairy roots could be selected from inoculated explants by screening root sections for GUS activity; this method eliminated the use of antibiotic resistance marker genes for selection. Transgenic hairy roots were produced from two cauliflower and four broccoli culivars. Shoots were regenerated from transgenic hairy root cultures of all four cultivars tested and successfully acclimatized to glasshouse conditions, although some plants had higher than diploid ploidy levels. Southern analysis confirmed the transgenic nature of these plants. T₀ plants from seven transgenic lines were crossed or selfed to produce viable seed. Genetic analysis of T₁ progeny confirmed the transmission of traits and revealed both independent and co-segregation of Ri T_L-DNA and vector T-DNA. GUS-positive phenotypically normal progeny free of T_L-DNA were identified in three transgenic lines out of the six tested representing all the cultivars regenerated including both cauliflower and broccoli.     

Production of transgenic Aralia elata regenerated from Agrobacterium rhizogenes-mediated transformed roots

Transgenic hairy roots were induced from petiole and root segments of in vitro plant Aralia elata, a medicinal woody shrub, after co-cultivation with A. rhizogenes ATCC 15834. The percentage of putative hairy root induction from root segments was higher (26.7%) than petiole explants (10.0%). Hairy roots showed active production of lateral roots with vigorous elongation. Transgenic plants were regenerated from hairy roots via somatic embryogenesis. These plants had wrinkled leaves, short petioles and numerous lateral hairy roots. The RT-PCR analysis showed the expression of rol A, B, C, D, aux 1 and 2 genes differed between the transgenic lines. Endogenous IAA level was higher in transgenic than non-transgenic plants. Conclusively, transgenic hairy roots were developed for first time in A. elata and the transgenic hairy root lines showed distinct morphological growth pattern and gene expression.     

Reactive oxygen species production and antioxidative defense in pea (Pisum sativum L.) root nodules after short-term aluminum treatment

Pea plants (Pisum sativum L.) were treated with 50???M aluminum chloride at pH 4.5 for 2 or 24?h at room temperature. Following treatment, root nodule Al uptake, the generation of reactive oxygen species (ROS, O ₂ ^?· and H₂O₂), and the activities of the antioxidant enzymes catalase (CAT), superoxide dismutase (SOD) and peroxidase (POX) were investigated. Aluminum accumulation was found chiefly in the apoplast of the nodule cortex, endodermis and meristem, while the formation of peroxide was detected in the nodule cortex, infection threads and bacteroidal tissue. Further, there were increased levels of superoxide in the meristem and bacteroidal tissue. The activity of SOD (EC 1.15.1.1) and POX (EC 1.11.1.7) increased in the Al-treated nodules and the roots of pea plants, whereas CAT (EC 1.11.1.6) activity decreased. The Al absorbed by the nodules induced ROS production. The POX and SOD are important ROS-scavengers in Al-stressed nodules.     

Flavonoid content and antioxidant activity of Artemisia vulgaris L. “hairy” roots

We investigated the effect of Agrobacterium rhizogenes-mediated transformation on antioxidant activity of Artemisia vulgaris “hairy” roots. It appeared that transformation may increase flavonoid content as well as DPPH-scavenging activity and ability to reduce Fe³⁺ as compared to the non-transformed plants. Some “hairy” roots accumulated flavonoids up to 73.1?±?10.6?mg RE/g DW (while the amount of flavonoids in the leaves of non-transformed plants was up to 49.4?±?5.0?mg RE/g DW). DPPH-scavenging activity of some “hairy” root lines was 3–3.8 times higher than such one of the roots of the control plants. The Fe³⁺-reducing power of most transgenic root extracts exceeded such power of the extracts of the roots of the control plants. The decrease in SOD activity was found in the most “hairy” root lines compared to the control roots. The increase of flavonoid content correlated with the increase of ability of extracts to scavenge DPPH*- radical and Fe³⁺ - reducing power. No correlation between SOD activity of extracts and concentration of flavonoids was found (p?≥?0.2).Thus, transformation has led to the alteration in flavonoid accumulation and antioxidant activity in A. vulgaris “hairy” roots. Transgenic roots with high-antioxidant properties can be selected after A. rhizogenes-mediated transformation.     

Involvement of Hydrogen Peroxide, Peroxidase and Superoxide Dismutase in Response of Medicago truncatula Lines Differing in Susceptibility to Phoma medicaginis Infection

This study was undertaken to assess the involvement of hydrogen peroxide (H₂O₂), peroxidase (POX; EC 1.11.1.7) and superoxide dismutase (SOD; EC 1.15.1.1) in Medicago truncatula in relation with susceptibility to Phoma medicaginis infection. Several M. truncatula lines were studied in terms of their response to P. medicaginis infection. Fifteen days after inoculation (dai), differences in susceptibility were found. DZA45.5 was the least susceptible line and F83005.5 was the most susceptible line. Microscopic analysis of fungal development was performed in inoculated detached leaves of the DZA45.5 and F83005.5 lines. No significant difference was observed in events from conidia germination to penetration. Differences became apparent during the colonization process as the pathogen was able to sporulate rapidly increasing its concentration on the tissue of F83005.5 in comparison with DZA45.5. To characterize the susceptibility of the two lines, histochemical detection of H₂O₂ was made in detached leaves. H₂O₂ detection showed an early accumulation of this component in cells of DZA45.5 at 1 dai. However, H₂O₂ was detected in few, if any, cells in the tissues of the most susceptible line, F83005.5. The activity of POX and SOD were determined spectrophotometrically in leaves of intact inoculated plants of both lines. Phoma medicaginis inoculation of DZA45.5 and F83005.5 did not affect POX activity level in leaves when compared with control uninoculated plants. SOD activity showed a significant decrease in F83005.5 and DZA45.5 leaves at 4 dai and 9 dai, respectively, in comparison with control plants. In control plants POX activity was significantly higher in the least susceptible line DZA45.5 in comparison with F83005.5. Early and higher production of H₂O₂ and elevated basal POX activity in cells of the least susceptible line, DZA45.5 could explain its ability to be less favourable to the colonization and reproduction of P. medicaginis in comparison with the most susceptible line, F83005.5.     

Induction of hairy roots and plant regeneration from the medicinal plant <Emphasis Type="Italic">Pogostemon Cablin</Emphasis>

An efficient transformation system for the medicinal and aromatic plant, Pogostemon cablin Benth was developed by using agropine-type Agrobacterium rhizogenes ATCC15834. Hairy roots formed directly from the cut edges of leaf explants or via callus stage 8 days after inoculation with the bacterium. The highest frequency of leaf explant transformation by Agrobacterium rhizogenes ATCC15834 was about 80% after infection for 25 days. Hairy roots grew rapidly on plant growth regulators (PGRs)-free Murashige and Skoog (MS) or 6,7-V medium and had characteristics of transformed roots such as fast growth and high lateral branching. The PCR amplification showed that rol genes of Ri plasmid of A. rhizogenes were integrated and expressed into the genome of transformed hairy roots. The hairy root line, PL6, grew very slowly in the first 8 days, then grew very quickly between day 8 and day 24. The optimum medium for callus induction of hairy roots consisted of 2.0 mg l⁻¹ benzyladenine (BA) and 0.1 mg/l α-naphthaleneacetic acid (NAA); while optimum medium for adventitious shoot regeneration from these cultures consisted of 0.1 mg l⁻¹ BA and 0.1 mg l⁻¹ NAA. Adventitious shoots could be rooted on 1/2MS. Southern blot analysis confirmed that rol genes of TL-DNA of Ri plasmid was integrated with at least three copies into the genome of hairy roots- regenerated P. cablin plants. The results presented provide a solid foundation for production of patchouli essential oil from hairy roots or its regenerated plants and also provide possibilities for utilization of artifical polyploidization or chemical mutation of hairy roots for improving germplasm and breeding of a new cultivar of P. cablin.     

Ri-plasmid as a helper for introducing vector DNA into alfalfa plants

Genetic engineering of legumes and other important dicotyledonous plants is limited because of the difficulty of regenerating plants via cell culture. Since a considerable number of crop plants can be regenerated only from root culture, the introduction of foreign genes into Agrobacterium rhizogenes-induced hairy roots may expand the list of crop plants that could be genetically engineered. Here we report genetic transformation of alfalfa (Medicago sativa L.), a valuable forage legume, using a virulent strain of Agrobacterium rhizogenes containing, in addition to its Ri-plasmid, a binary vector containing a nopaline synthase gene. Plant cells transformed by this vector can be easily identified by their ability to produce nopaline. Transformed alfalfa plants were recovered from A. rhizogenes-induced hairy roots. These transgenic plants were characterized by normal leaf morphology and stem growth but a root system that was shallow and more extensive than normal. These plants were also fertile, set seeds upon self-pollination and outcrossing. Nopaline was detected in R1 progeny. Southern blot analysis confirmed the presence of multiple copies of T-DNAs from the Riplasmid in the plant genome in addition to the vector T-DNA.     

Antioxidative defense system in pigeonpea roots under waterlogging stress

Pigeonpea [Cajanus cajan (L.) Millsp.] is a waterlogging-sensitive legume crop. We studied the effect of waterlogging stress on hydrogen peroxide (H₂O₂) content, lipid peroxidation and antioxidant enzyme activities in two pigeonpea genotypes viz., ICPL-84023 (waterlogging resistant) and MAL-18 (waterlogging susceptible). In a pot experiment, waterlogging stress was imposed for 6 days at early vegetative stage (20 days after sowing). Waterlogging treatment significantly increased hydrogen peroxide accumulation and lipid peroxidation, which indicated the extent of oxidative injury posed by stress conditions. Enzyme activities of peroxidase (POX), catalase (CAT), ascorbate peroxidase (APX), superoxide dismutase (SOD) and polyphenol oxidase (PPO) increased in pigeonpea roots as a consequence of waterlogged conditions, and all the enzyme activities were significantly higher in waterlogged ICPL-84023 than in MAL-18. POX activity was the maximum immediately after imposing stress, therefore, it was suggested to be involved in early scavenging of H₂O₂, while rest of the enzymes (CAT, APX, SOD and PPO) were more important in late responses to waterlogging. Present study revealed that H₂O₂ content is directly related to lipid peroxidation leading to oxidative damage during waterlogging in pigeonpea. Higher antioxidant potential in ICPL-84023 as evidenced by enhanced POX, CAT, APX, SOD and PPO activities increased capacity for reactive oxygen species (ROS) scavenging and indicated relationship between waterlogging resistance and antioxidant defense system in pigeonpea.     

Overexpression of the Arabidopsis thaliana squalene synthase gene in Withania coagulans hairy root cultures

Squalene synthase (SS) dimerizes two molecules of farnesyl diphosphate to synthesize squalene, a shared precursor in steroid and triterpenoid biosynthesis in plants. The SS1 gene encoding SS from Arabidopsis thaliana was introduced in Withania coagulans under the control of the CaMV35S promoter together with the T-DNA of Agrobacterium rhizogenes A4. The engineered hairy roots were studied for withanolide production and phytosterol accumulation and the results were compared with those obtained from control roots harbouring only the T-DNA from pRiA4. The increased capacity of the engineered roots for biosynthesizing phytosterols and withanolides was strongly related with the expression level of the transgene, showing the effectiveness of overexpressing 35SS1 to increase triterpenoid biosynthesis.     

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 (Km^R) 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 Km^R 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. Received: 11 August 1998 / Revision received: 17 February 1999 / Accepted: 18 March 1999     

Cross-talks between Ca<Superscript>2+</Superscript>/CaM and H<Subscript>2</Subscript>O<Subscript>2</Subscript> in abscisic acid-induced antioxidant defense in leaves of maize plants exposed to water stress

Here we examined whether Ca²⁺/Calmodulin (CaM) is involved in abscisic acid (ABA)-induced antioxidant defense and the possible relationship between CaM and H₂O₂ in ABA signaling in leaves of maize (Zea mays L.) plants exposed to water stress. An ABA-deficient mutant vp5 and its wild type were used for the experimentation. We found that water stress enhanced significantly the contents of CaM and H₂O₂, and the activities of chloroplastic and cytosolic superoxide dismutase (SOD), ascorbate peroxidase (APX) and glutathione reductase (GR), and the gene expressions of the CaM1, cAPX, GR1 and SOD4 in leaves of wild-type maize. However, the increases mentioned above were almost arrested in vp5 plants and in the wild-type plants pretreated with ABA biosynthesis inhibitor tungstate (T), suggesting that ABA is required for water stress-induced H₂O₂ production, the enhancement of CaM content and antioxidant defense. Besides, we showed that the up-regulation of water stress-induced antioxidant defense was almost completely blocked by pretreatment with Ca²⁺ inhibitors, CaM antagonists and reactive oxygen (ROS) manipulators. Moreover, the analysis of time course of CaM and H₂O₂ production under water stress showed that the increase in CaM content preceded that of H₂O₂. These results suggested that Ca²⁺/CaM and H₂O₂ were involved in the ABA-induced antioxidant defense under water stress, and the increases of Ca²⁺/CaM contents triggered H₂O₂ production, which inversely affected the contents of CaM. Thus, a cross-talk between Ca²⁺/CaM and H₂O₂ may play a pivotal role in the ABA signaling.     

Shoot and root culture of Hypericum perforatum L. transformed with Agrobacterium rhizogenes A4M70GUS

Hairy root cultures of Hypericum perforatum were obtained following inoculation of aseptically germinated seedlings with A. rhizogenes strain A4M70GUS. Effect of sucrose on the growth and biomass production of hairy root cultures was investigated. Hairy root cultures spontaneously regenerated shoots buds from which a number of shoot culture clones was established. Transformed shoot cultures exhibited good shoot multiplication, elongation and rooting on a hormone-free woody plant medium. Plants regenerated from hairy roots were similar in appearance to the normal, nontransformed plants.     

Effect of salinity and waterlogging on growth,anatomical and antioxidative responses in <Emphasis Type="Italic">Mentha aquatica</Emphasis> L.

Salinity and waterlogging are two stresses which in nature often occur simultaneously. In this work, effects of combined waterlogging and salinity stresses are studied on the anatomical alteration, changes of enzymatic antioxidant system and lipid peroxidation in Mentha aquatica L. plants. Seedlings were cultured in half-strength Hoagland medium 50 days after sowing, and were treated under combination of three waterlogging levels (well drained, moderately drained and waterlogging) and NaCl (0, 50, 100, 150 mM) for 30 days. Moderately drained and waterlogging conditions induced differently aerenchyma formation in roots of M. aquatica salt-treated and untreated plants. Moreover, stele diameter and endodermis layer were also affected by salt stress and waterlogging. Salt stress significantly decreased growth, relative water content (RWC), protein level, catalase (CAT) and polyphenol oxidase (PPO) activities, and increased proline content, MDA content, H₂O₂ level and activities of superoxide dismutase (SOD), peroxidase (POX), and ascorbate peroxidase (APX). Waterlogging in salt-untreated plants increased significantly growth parameters, RWC, protein content, antioxidant enzyme activity, and decreased proline content, H₂O₂ and MDA levels. In salt-treated plant, waterlogging caused strong induction of antioxidant enzymes activities especially at severe stress condition. These results suggest M. aquatica is a waterlogging tolerant plant due to significant increase of antioxidant activity, membrane stability and growth under water stress. High antioxidant capacity under waterlogging can be a protective strategy against oxidative damage, and help to salt stress alleviation.