Hairy Root System Research Articles

Root in vitro cultures of six Gypsophila species and their saponin contents

A simple and rapid method of excised root cultures from six Gypsophila species was performed allowing continuous growth without phytohormones. Established on MH3 medium from solid-grown seedlings these roots were subcultured for 1 year on a solid medium before being transferred in a liquid medium to obtain substantial biomass for saponin content analysis. Morphologically, the different root lines presented different growth behaviors and different physical aspects: some have linear growth by the tip of the main axial root from the original seedling; others have additional lateral root initiations producing a hairy root-system more or less dense. A marked increase of biomass was observed in the light by comparison with dark conditions. Significant growth for Gypsophila glomerata was achieved within 3 weeks on liquid medium; biomass grew up to 50-fold in batch cultures reaching 10 g DW. The fingerprints of the saponin HPLC profiles of the six Gypsophila species were drastically different with at least up to 30 different saponins detected for some of them. The roots of Gypsophila elegans accumulated saponins up to 65 mg/g DW. These amounts were higher than in Gypsophila paniculata roots classically found as the best producing ones. On the contrary the root lines of G. glomerata showed a smaller quantitative amount of saponins (between 1.3 and 7.10 mg/g DW) than those of G. elegans but nearly the same HPLC profiles as for root extracts of G. paniculata plants grown directly in the fields.

Establishment of a transgenic hairy root system in wild and domesticated watermelon (Citrullus lanatus) for studying root vigor under drought

Root vigor is an important trait for the growth of terrestrial plants, especially in water-deficit environments. Although deserts plants are known for their highly developed root architecture, the molecular mechanism responsible for this trait has not been determined. Here we established an efficient protocol for the genetic manipulation of two varieties of watermelon plants: a desert-grown wild watermelon that shows vigorous root growth under drought, and a domesticated cultivar showing retardation of root growth under drought stress. Agrobacterium rhizogenes-mediated transgenic hairy roots were efficiently induced and selected from the hypocotyls of these plants. Transgenic GUS expression was detected in the roots by RT-PCR and histochemical GUS staining. Moreover, a liquid culture system for evaluating their root growth was also established. Interestingly, growth of the hairy roots derived from domesticated variety of watermelon strongly inhibited under high osmotic condition, whereas the hairy roots derived from wild variety of watermelon retained substantial growth rates under the stress condition. The new protocol presented here offers a powerful tool for the comparative study of the molecular mechanism underlying drought-induced root growth in desert plants.

Rapid in planta evaluation of root expressed transgenes in chimeric soybean plants

Production of stable transgenic plants is one factor that limits rapid evaluation of tissue specific transgene expression. To hasten the assessment of transgenes in planta, we evaluated the use of chimeric soybean seedlings expressing transgenic products in roots. Tap roots from four-day old seedlings (cultivars 'Jack' and KS4704) were excised and hairy roots were induced from hypocotyls via Agrobacterium rhizogenes-mediated transformation. Inoculated hypocotyls were screened on a MS-based medium containing either 200 mg/L kanamycin or 20 mg/L hygromycin. Beta-glucuronidase (GUS) activity assay indicated that highest GUS expression was observed in hypocotyls exposed to a 4-d pre-inoculation time, a neutral pH (7.0) for the co-cultivation medium. A 170-bp of the Fib-1 gene and 292-bp of the Y25C1A.5 gene fragments, both related to nematode reproduction and fitness, were cloned independently into pANDA35HK vector using a Gateway cloning strategy. The resulting RNAi constructs of the genes fragments were transformed into soybean using the chimeric hairy root system and evaluated for its effect on soybean cyst nematode (Heterodera glycines) fecundity. Confirmation of transformation was attained by polymerase chain reaction and Southern-blot analysis, and some potential for suppression of H. glycines reproduction was detected for the two constructs. This method takes on average four weeks to produce chimeric plants ready for transgene analysis.

Biochemical and molecular characterization of PvPAP3, a novel purple acid phosphatase isolated from common bean enhancing extracellular ATP utilization.

Purple acid phosphatases (PAPs) play diverse physiological roles in plants. In this study, we purified a novel PAP, PvPAP3, from the roots of common bean (Phaseolus vulgaris) grown under phosphate (Pi) starvation. PvPAP3 was identified as a 34-kD monomer acting on the specific substrate, ATP, with a broad pH range and a high heat stability. The activity of PvPAP3 was insensitive to tartrate, indicating that PvPAP3 is a PAP-like protein. Amino acid sequence alignment and phylogenetic analysis suggest that PvPAP3 belongs to the group of plant PAPs with low molecular mass. Transient expression of 35S:PvPAP3-green fluorescent protein in onion (Allium cepa) epidermal cells verified that it might anchor on plasma membrane and be secreted into apoplast. Pi starvation led to induction of PvPAP3 expression in both leaves and roots of common bean, and expression of PvPAP3 was strictly dependent on phosphorus (P) availability and duration of Pi starvation. Furthermore, induction of PvPAP3 expression was more rapid and higher in a P-efficient genotype, G19833, than in a P-inefficient genotype, DOR364, suggesting possible roles of PvPAP3 in P efficiency in bean. In vivo analysis using a transgenic hairy root system of common bean showed that both growth and P uptake of bean hairy roots from the PvPAP3 overexpression transgenic lines were significantly enhanced when ATP was supplied as the sole external P source. Taken together, our results suggest that PvPAP3 is a novel PAP that might function in the adaptation of common bean to P deficiency, possibly through enhancing utilization of extracellular ATP as a P source.

Mass production and application of activation tagged hairy root lines for functional genomic of secondary metabolism in ginseng

Activation tagging that uses T-DNA vectors containing multimerized transcriptional enhancers from the cauliflower mosaic virus (CaMV) 35S gene is a powerful tool to determine gene function in plants. This approach has been successfully applied in screening various types of mutations and cloning the corresponding genes. We generated an activation tagged hairy root pool of ginseng (Panax ginseng C.A. Meyer) in an attempt to isolate genes involved in the biosynthetic pathway of ginsenoside (triterpene saponin), which is known as the major active ingredient of the root. Quantitative and qualitative variation of ginsenoside in activation tagged hairy root lines were profiled using LC/MS. Metabolic profiling data enabled selection of a specific hairy root line which accumulated ginsenoside at a higher level than other lines. The relative expression level of several genes of triterpene biosynthetic pathway in the selected hairy root line was determined by real time RT-PCR. Overall results suggest that the activation tagged ginseng hairy root system described in this study would be useful in isolating genes involved in a complex metabolic pathway from genetically intractable plant species by metabolic profiling.

Establishment of transgenic tobacco hairy roots expressing basic peroxidases and its application for phenol removal

Transgenic hairy root (HR) systems constitute an interesting alternative to improve the efficiency of phytoremediation process. Since peroxidases (Px) have been associated with phenolic compounds removal, in the present work, transgenic tobacco HR, which expressed basic Px genes from tomato ( tpx1 and tpx2), were established and assayed for phenol removal. Tobacco HR clones were obtained, including those transgenic for TPX1 or TPX2, those double transgenic (DT) for both Px and the corresponding controls. Based on growth index, the presence of rol C sequence, tpx1 and/or tpx2 genes and the coded proteins, as well as Px activity determinations, we selected 10 tobacco HR clones for phenol removal assays. The removal efficiencies were high for all the HR, although, some transgenic HR showed significantly higher removal efficiencies compared with controls. The results demonstrate that TPX1 is involved in phenol removal not only when it was overexpressed in tomato, but also when it was expressed in other plant, such as tobacco. The higher efficiency of TPX2 transgenic HR showed that this Px also participates in the process. The contribution of other mechanisms (adsorption, H 2O 2 independent enzymatic processes) could be considered depreciable, which establishes the great implication of Px in phenol removal.

Flux quantification in central carbon metabolism of Catharanthus roseus hairy roots by 13C labeling and comprehensive bondomer balancing

Methods for accurate and efficient quantification of metabolic fluxes are desirable in plant metabolic engineering and systems biology. Toward this objective, we introduce the application of “bondomers”, a computationally efficient and intuitively appealing alternative to the commonly used isotopomer concept, to flux evaluation in plants, by using Catharanthus roseus hairy roots as a model system. We cultured the hairy roots on (5% w/w U– 13C, 95% w/w naturally abundant) sucrose, and acquired two-dimensional [ 13C, 1H] and [ 1H, 1H] NMR spectra of hydrolyzed aqueous extract from the hairy roots. Analysis of these spectra yielded a data set of 116 bondomers of β-glucans and proteinogenic amino acids from the hairy roots. Fluxes were evaluated from the bondomer data by using comprehensive bondomer balancing. We identified most fluxes in a three-compartmental model of central carbon metabolism with good precision. We observed parallel pentose phosphate pathways in the cytosol and the plastid with significantly different fluxes. The anaplerotic fluxes between phosphoenolpyruvate and oxaloacetate in the cytosol and between malate and pyruvate in the mitochondrion were relatively high (60.1 ± 2.5 mol per 100 mol sucrose uptake, or 22.5 ± 0.5 mol per 100 mol mitochondrial pyruvate dehydrogenase flux). The development of a comprehensive flux analysis tool for this plant hairy root system is expected to be valuable in assessing the metabolic impact of genetic or environmental changes, and this methodology can be extended to other plant systems.

Potential for rhizofiltration of uranium using hairy root cultures of Brassica juncea and Chenopodium amaranticolor

Hairy root cultures of Brassica juncea and Chenopodium amaranticolor were developed by genetic transformation using Agrobacterium rhizogenes. The stable, transformed root systems demonstrated a high growth rate of 1.5–3.0g/g dry weight/day in Murashige and Skoog medium. In the present study, hairy root system was used for removal of uranium from the solution of concentration up to 5000μM. The results indicated that the hairy roots could remove uranium from the aqueous solution within a short period of incubation. B. juncea could take up 20–23% of uranium from the solution containing up to 5000μM, when calculated on g/g dry weight basis. C. amaranticolor showed a slow and steady trend in taking up uranium, with 13% uptake from the solution of 5000μM concentration. Root growth was not affected up to 500μM of uranium nitrate over a period of 10 days.

Large-scale plant micropropagation.

Plant micropropagation is an efficient method of propagating disease-free, genetically uniform and massive amounts of plants in vitro. The micropropagation from cells can be achieved by direct organogenesis from hairy roots or regeneration via somatic tissue. Once the availability of embryogenic cell and hairy root systems based on liquid media has been demonstrated, the scale-up of the whole process should be established by an economically feasible technology for their large-scale production in appropriate bioreactors. It is necessary to design a suitable bioreactor configuration that can provide adequate mixing and mass transfer while minimizing the intensity of shear stress and hydrodynamic pressure. Automatic selection of embryogenic calli and regenerated plantlets using an image analysis procedure should be associated with the system. Using the above systems, it will be possible to establish an advanced plant micropropagation system in which the plantlets can be propagated without soil under optimal conditions controlled in plant factory. The aim of this review is to identify the problems related to large-scale plant micropropagation via somatic embryogenesis and hairy roots, and to summarize the most recent developments in bioreactor design. Emphasis is placed on micropropagation technology and computer-aided image analysis, including the successful results obtained in our laboratories.

Root hairiness: effect on fluid flow and oxygen transfer in hairy root cultures

The effect of root hairiness on fluid flow and oxygen transfer in hairy root cultures was investigated using wild-type, transgenic and root-hair mutants of Arabidopsis thaliana. The root hair morphologies of the A. thaliana lines were hairless, short hairs, moderately hairy (wild-type) and excessively hairy, and these morphologies were maintained after transformation of seedlings with Agrobacterium rhizogenes. Filtration experiments were used to determine the permeability of packed beds of roots; permeability declined significantly with increasing root hairiness as well as with increasing biomass density. Hairy roots of wild-type A. thaliana grew fastest with a doubling time of 6.9 days, but the hairless roots exhibited the highest specific oxygen uptake rate. In experiments using a gradientless packed bed reactor with medium recirculation, the liquid velocity required to eliminate external mass transfer boundary layer effects increased with increasing root hairiness, reflecting the greater tendency towards liquid stagnation near the surface of roots covered with hairs. External critical oxygen tensions also increased with increasing root hairiness, ranging from 50% air saturation for hairless roots to ca. 150% air saturation for roots with excessive root hairs. These results are consistent with root hairs providing a significant additional resistance to oxygen transfer to the roots, indicating that very hairy roots are more likely than hairless roots to become oxygen-limited in culture. This investigation demonstrates that root hairiness is an important biological parameter affecting the performance of root cultures and suggests that control over root hair formation, either by use of genetically modified plant lines or manipulation of culture conditions, is desirable in large-scale hairy root systems.

Expression of Soybean Cyst Nematode Resistance in Transgenic Hairy Roots of Soybean

ABSTRACTTransgenic hairy roots of soybean [Glycine max (L.) Merrill] induced by Agrobacterium rhizogenes support the complete life cycle of soybean cyst nematode (SCN, Heterodera glycines Ichinohe) in vitro. However, expression of SCN resistance in hairy soybean roots has not been investigated. A transgenic hairy root system would be useful in developing an assay for candidate SCN resistance genes. The objectives of this study were to characterize transgene expression in SCN‐infected hairy soybean roots and to evaluate a transgenic hairy root system for investigations of resistance to SCN. Seedling cotyledons of the SCN‐susceptible cultivars, Agassiz and Parker, and SCN‐resistant Bell and Faribault were infected with A. rhizogenes strain K599 transformed with T‐DNA binary vectors containing the gusA gene fused to promoters from either the cauliflower mosaic virus (CaMV 35S), Arabidopsis thaliana phenylalanine ammonia lyase (PAL), or bean (Phaseolus vulgaris L.) chalcone synthase‐8 (CHS) genes. Nine days after inoculating transgenic hairy roots with sterile J2 nematodes, CHS‐regulated β‐glucuronidase (GUS) staining at infection sites increased in hairy roots of resistant Faribault and decreased in susceptible Agassiz. PAL‐regulated GUS staining was absent at infection sites in hairy roots of resistant cultivars, but was increased in infection sites in susceptible cultivars. Thirty‐five days after inoculation with SCN, the mean number of cysts formed on hairy roots of the resistant cultivars was about 14% of the mean number of cysts formed on hairy roots of the susceptible cultivars, indicating that the SCN resistance phenotypes were preserved in transgenic hairy roots. These results indicated that the transgenic hairy soybean root system will be useful for investigating differential transgene expression during nematode infection and evaluation of candidate SCN resistance genes.

Stable transformation of Lithospermum erythrorhizon by Agrobacterium rhizogenes and shikonin production of the transformants.

Seedling hypocotyls of Lithospermum erythrorhizon were infected with Agrobacterium rhizogenes (strain 15834) harboring a binary vector with an intron-bearing the β-glucuronidase (GUS) gene driven by cauliflower mosaic virus (CaMV) 35S promoter as well as the hygromycin phosphotransferase (HPT) gene as the selection marker. About 20% of the hairy roots isolated were hygromycin resistant and had co-integrated GUS and HPT genes in their Lithospermum genomic DNA. Because GUS activity was detected in almost all the hygromycin-resistant root tissues, the CaMV 35S promoter seems to be ubiquitously active in L. erythrorhizon hairy roots. In pigment production medium M9, the hairy root cultures had shikonin productivity similar to that of cell suspension cultures of Lithospermum. They also showed light-dependent inhibition of shikonin biosynthesis similar to that of Lithospermum cell cultures. These findings suggest that this hairy root system transformable with A. rhizogenes is a suitable model system for molecular characterization of shikonin biosynthesis via reverse genetics.

High frequency transformation and regeneration of transgenic plants in the model legumeLotus japonicus

The molecular analysis of plant genes involved in nodulation has been slowed by the inability to produce high numbers of transgenic legume lines. The high efficiency gene transfer and plant regeneration systems of the model legume Lotus japonicus is described. A collection of wild-type A. rhizogenes strains was tested for infectivity and the most virulent strains, 9402 and AR10, were selected for further use. Growth conditions for plantlets, induction of hairy roots and nodulation of composite plants were optimized for large-scale screening in Petri dishes. A cluster of 3-10 nodules was regularly formed on transgenic hairy roots 7-12 d after inoculation with the effective Rhizobium loti strain NZP2235. There were no apparent morphological differences between nodulation of hairy and wildtype roots. To test the applicability of the hairy root system for the trapping of symbiotic genes, transformation experiments with binary vectors possessing a 8-g/ucuronidas e {gus, uidA) or a luciferase [luc) reporter driven by a cauliflower mosaic virus (CaMV) 35S promoter were performed. The frequency of cotransfer of a binary T-DNA with a root-inducing (Ri) T-DNA was 70%. Positive expression suggests that gus and luc trap vectors can be used for gene tagging in L. japonicus. To open the possibility of searching for mutant phenotypes, a regeneration system has been developed enabling the regeneration of large numbers of transgenic plants from hairy root cultures in about 5-6 months. At the same time, the A. tumefaciens hypocotyl transformation regeneration in L. japonicus has been improved. This new version provides fertile transgenic plants in about 4 months.

Conservation of T-DNA in Plants Regenerated from Hairy Root Cultures

T1-plants (first generation of transformed plants) regenerated from hairy root culture lines synthesized the same opines and carried T-DNA with the same restriction pattern as the parental line. Opine synthesis segregated in the progeny of T1-plants (T2-plants), Two opine-positive T2-plants had essentially the same T-DNA structure as the parental T1-plant, Clonal constitution of hairy root cultures, facility of regeneration, stability of opine synthesis, and T-DNA structure appear to be valuable attributes of the hairy root system for studies on genetic engineering of plants.