Ara-Rhizotron: An Effective Culture System to Study Simultaneously Root and Shoot Development of Arabidopsis

Studying Arabidopsis thaliana (L.) Heynh. root development in situ at the whole plant level without affecting shoot development has always been a challenge. Such studies are usually carried out on individual plants, neglecting competition of a plant population, using hydroponic systems or Agar-filled Petri dishes. Those both systems, however, present some limitations, such as difficulty to study precisely root morphogenesis or time-limited culture period, respectively. In this paper, we present a method of Arabidopsis thaliana (L.) Heynh. cultivation in soil medium, named “Ara-rhizotron”. It allows the non-destructive study of shoot and root development simultaneously during the entire period of vegetative growth. In this system, roots are grown in 2D conditions, comparable to other soil cultures. Moreover, grouping several Ara-rhizotrons in a box enables the establishment of 3D shoot competition as for plants grown in a population. In comparison to a control culture grown in pots in the same environmental conditions, the Ara-rhizotron resulted in comparable shoot development in terms of dry mass, leaf area, number of leaves and nitrogen content. We used this new culture system to study the effect of irrigation modalities on plant development. We found that irrigation frequency only affected root partitioning in the soil and shoot nitrogen content, but not shoot or root growth. These effects appeared at the end of the vegetative growth period. This experiment highlights the opportunity offered by the Ara-rhizotron to point out tardy effects, affecting simultaneously shoot development and root architecture of plants grown in a population. We discuss its advantages in relation to root development and physiology, as well as its possible applications.     

Zoosporangial root infection of tomato by Spongospora subterranea in hydroponic and glasshouse culture results in diminished plant growth

Tomato plants are highly susceptible to root infection by Spongospora subterranea and are commonly used as bioassay hosts. The impacts of root infection with S. subterranea on plant productivity and yield have been debated. Recent experiments with potato, the major economic host of S. subterranea, have indicated significantly reduced plant growth and potato yield following heavy infection. However, there have been very few similar studies that have examined the possible impacts of S. subterranea infection on tomato plant growth. Three tomato cultivars, “Grape,” “Roma” and “Truss,” were challenged with S. subterranea inoculum in hydroponic culture. Moderate to severe zoosporangial infections were observed with minor but statistically significant differences in susceptibility among the three tomato cultivars. Zoosporangial root infection in the absence of root gall formation resulted in significantly diminished shoot lengths and plant fresh weights in pathogen challenge tests conducted both in hydroponic culture and glasshouse‐grown plants in potting mix. Root lengths were reduced, but the differences were statistically significant in a single trial only. The findings from this study demonstrate that, as with potato, root infection by S. subterranea can result in reduced tomato plant growth and that root gall production associated with root infection was not necessary for this retardation of growth response. This further suggests that possible yield impacts in other crop species that are hosts for S. subterranea root infection are worthy of examination.     

<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.     

Compared root system architectures in seedlings and in vitro plantlets of Hevea brasiliensis,in the initial years of growth in the field

In vitro culture of Hevea was undertaken to propagate selected clones on their own roots. The challenge was to overcome the failure of cuttings due to the poor conformity of regenerated root systems. Trees of several juvenile or mature genotypes were propagated either by in vitro microcutting, or by somatic embryogenesis, and planted in the field. Certain static and dynamic components of the root system were observed at different growth stages, from 0 to 3 years, and compared to those of seedlings of the same age used in the trial as a reference. A simple method was designed for measuring the vigour and balance of the root system. The in vitro plantlets had a well-developed taproot and lateral root system, with an architecture similar to that of plants obtained from seed. Moreover, clear differences occurred between selected clones for the relative vigour of the tap roots, lateral roots and trunk. This revised version was published online in June 2006 with corrections to the Cover Date.     

二花蝴蝶草的组织培养及植株再生

二花蝴蝶草(Torenia biniflora)为玄参科蝴蝶草属一年生植物,分布于广东、广西等亚热带地区,是一种观赏性较高的野生花卉。在自然生长条件下,二花蝴蝶草繁殖速度慢、增殖率低,而且花色和花型种类偏少,无法满足市场多样化的要求。植物组织培养技术为观赏植物的品种改良和新品种选育提供了新途径,目前蓝猪耳、蔓性蝴蝶草和单色蝴蝶草等蝴蝶草属植物的组织培养已获得成功,但二花蝴蝶草的组织培养尚未见有相关报道。该研究以二花蝴蝶草全展叶片为外植体,研究了培养基中添加不同种类和浓度植物生长物质对不定芽诱导和生长的影响,以及离子强度和不同浓度IBA对生根的影响。根据不定芽的诱导率和平均芽数筛选出最佳不定芽诱导培养基,并从生根率、平均根数和平均根长等方面筛选出最佳生根培养基。结果表明:不定芽诱导与植物生长物质的浓度和种类有关,以MS+6-BA 0.5 mg·L-1+NAA 0.2 mg·L-1培养基的诱导效果最佳;二花蝴蝶草生根的最佳基本培养基为1/2MS,不同浓度的IBA对二花蝴蝶草的生根影响也不相同,其中以IBA(0.05 mg·L-1)诱导不定芽的生根效果最佳。该研究建立了二花蝴蝶草的高频离体再生体系,为二花蝴蝶草的快速繁殖和遗传转化研究奠定了基础。     

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.     

Interactions between the soilborne root pathogenPhytophthora nicotianae var.parasitica and the arbuscular mycorrhizal fungusGlomus mosseae in tomato plants

In order to study the influence of Arbuscular Mycorrhiza (AM) on the development of root rot infection, tomato plants were raised with or withoutGlomus mosseae and/orPhytophthora nicotianae var.parasitica in a sand culture system. All plants were fed with a nutrient solution containing one of two phosphorus (P) levels, 32µM (I P) or 96µM (II P), to test the consequence of enhanced P nutrition by the AM fungus on disease dynamics. Mycorrhizal plants had a similar development to that of control plants. Treatment withPhytophthora nicotianae var.parasitica resulted in a visible reduction in plant weight and in a widespread root necrosis in plants without mycorrhiza. The presence of the AM fungus decreased both weight reduction and root necrosis. The percentage reduction of adventitious root necrosis and of necrotic root apices ranged between 63 and 89% The enhancement of P nutrition increased plant development, but did not appreciably decrease disease spread. In our system, mycorrhiza increased plant resistance toP. nicotianae var.parasitica infection. Although a contribution of P nutrition by mycorrhiza cannot be excluded, other mechanisms appear to play a crucial role.     

Interaction of Glomus mosseae and Paecilomyces lilacinus on Meloidogyne javanica of tomato

The effects of Glomus mosseae and Paecilomyces lilacinus on Meloidogyne javanica of tomato were tested in a greenhouse experiment. Chicken layer manure was used as a carrier substrate for the inoculum of P. lilacinus. The following parameters were used: gall index, average number of galls per root system, plant height, shoot and root weights. Inoculation of tomato plants with G. mosseae did not markedly increase the growth of infected plants with M. javanica. Inoculation of plants with G. mosseae and P. lilacinus together or separately resulted in similar shoots and plant heights. The highest root development was achieved when mycorrhizal plants were inoculated with P. lilacinus to control root-knot nematode. Inoculation of tomato plants with G. mosseae suppressed gall index and the average number of galls per root system by 52% and 66%, respectively, compared with seedlings inoculated with M. javanica alone. Biological control with both G. mosseae and P. lilacinus together or separately in the presence of layer manure completely inhibited root infection with M. javanica. Mycorrhizal colonization was not affected by the layer manure treatment or by root inoculation with P. lilacinus. Addition of layer manure had a beneficial effect on plant growth and reduced M. javanica infection.     

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     

Effect of fluoride supply on fluoride concentrations in five pasture species: Levels required to reach phytotoxic or potentially zootoxic concentrations in plant tissue

Recent findings have highlighted the possibility of increased fluoride (F) concentrations in herbage through F taken up from soil via the plant root. This paper aimed to assess the risk of F concentrations reaching phytotoxic or zootoxic concentrations in pasture plants. Five plant species commonly found in improved pastures in Australia, the sown species subterranean clover (Trifolium subterranean) and cocksfoot (Dactylis glomerata), and weeds barley grass (Hordeum leporinum), scotch thistle (Onopordum acanthium) and sorrel (Rumex acetosella) were grown in complete nutrient solutions with graded levels of added F to determine the effects of F⁻ activity in solution on phytotoxicity and uptake of F by their roots. A model was developed using data from these solution culture experiments and data from the literature. The model assessed uptake of F by plants grown over a range of soil pH values and determined the risk of F taken up through the plant roots reaching phytotoxic concentrations, or concentrations potentially injurious to grazing animals, in the plant shoots. Modelling data suggested that the plants studied would not accumulate phytotoxic concentrations of F in shoots or concentrations of F deleterious to grazing animals through root uptake in neutral pH agricultural soils. The risks from F addition to soils in phosphatic fertilisers leading to reduction in pasture growth or animal health are therefore low. However, in highly F-polluted soil, as the soil becomes more acidic or alkaline, the risk of zootoxic concentrations of F in shoots of plants would increase. This revised version was published online in June 2006 with corrections to the Cover Date.     

Arbuscular mycorrhizal fungi reduce growth and infect roots of the non‐host plant Arabidopsis thaliana

The arbuscular mycorrhizal (AM) symbiosis is widespread throughout the plant kingdom and important for plant nutrition and ecosystem functioning. Nonetheless, most terrestrial ecosystems also contain a considerable number of non‐mycorrhizal plants. The interaction of such non‐host plants with AM fungi (AMF) is still poorly understood. Here, in three complementary experiments, we investigated whether the non‐mycorrhizal plant Arabidopsis thaliana, the model organism for plant molecular biology and genetics, interacts with AMF. We grew A. thaliana alone or together with a mycorrhizal host species (either Trifolium pratense or Lolium multiflorum) in the presence or absence of the AMF Rhizophagus irregularis. Plants were grown in a dual‐compartment system with a hyphal mesh separating roots of A. thaliana from roots of the host species, avoiding direct root competition. The host plants in the system ensured the presence of an active AM fungal network. AM fungal networks caused growth depressions in A. thaliana of more than 50% which were not observed in the absence of host plants. Microscopy analyses revealed that R. irregularis supported by a host plant was capable of infecting A. thaliana root tissues (up to 43% of root length colonized), but no arbuscules were observed. The results reveal high susceptibility of A. thaliana to R. irregularis, suggesting that A. thaliana is a suitable model plant to study non‐host/AMF interactions and the biological basis of AM incompatibility.     

Root growth of wetland plants with different root types

A wastewater culture system was designed to study the root growth of eight species of wetland plants with two different root types. The system included a plastic barrel for holding the wastewater and a foam plate for holding the plant. The results indicated that the root growth of the plants with fibril roots was faster than that of the plants with rhizomatic roots. The species with fibril roots had higher root number (1349 per plant) than species with rhizomatic roots (549 per plant) after ten weeks of cultivation. The average root biomass of plants with fibril roots was 11.3 g per plant, whereas that of plants with rhizomatic roots was 7.4 g per plant. Fine root biomass of diameter ≤ 1 mm constituted 51.9% of the total root biomass in plants with fibril roots, whereas it accounted for only 25.1% in plants with rhizomatic roots. The root surface area of the plants with fibril roots (6933 cm² per plant) was markedly larger than that of the species with rhizomatic roots (1897 cm² per plant). The species with rhizomatic roots showed a longer root lifespan (46.6 days) than those with fibril roots (34.8 days).     

Stimulation of root hair elongation in Arabidopsis thaliana by low phosphorus availability

Low phosphorus availability stimulates root hair elongation in many plants, which may have adaptive significance in soil phosphorus acquisition. We investigated the effect of low phosphorus on the elongation of Arabidopsis thaliana root hairs. Arabidopsis thaliana plants were grown in plant culture containing high (1000 mmol m^?3) or low (1 mmol m^?3) phosphorus concentrations, and root hair elongation was analysed by image analysis. After 15d of growth, low-phosphorus plants developed root hairs averaging 0.9 mm in length while high-phosphorus plants of the same age developed root hairs averaging 0.3 mm in length. Increased root hair length in low-phosphorus plants was a result of both increased growth duration and increased growth rate. Root hair length decreased logarithmically in response to increasing phosphorus concentration. Local changes in phosphorus availability influenced root hair growth regardless of the phosphorus status of the plant. Low phosphorus stimulated root hair elongation in the hairless axr2 mutant, exogenously applied IAA stimulated root hair elongation in wild-type high-phosphorus plants and the auxin antagonist CM PA inhibited root hair elongation in low-phosphorus plants. These results indicate that auxin may be involved in the low-phosphorus response in root hairs.     

Contaminant Removal of Domestic Wastewater by Constructed Wetlands： Effects of Plant Species

A comparative study of the efficiency of contaminant removal between five emergent plant species and between vegetated and unvegetated wetlands was conducted in small-scale （2.0 m×1.0 m×0.7 m, lengthxwidthxdepth） constructed wetlands for domestic wastewater treatment in order to evaluate the decontaminated effects of different wetland plants. There was generally a significant difference in the removal of total nitrogen （TN） and total phosphorus （TP）, but no significant difference in the removal of organic matter between vegetated and unvegetated wetlands. Wetlands planted with Canna indica Linn., Pennisetum purpureum Schum., and Phragmites communis Trin. had generally higher removal rates for TN and TP than wetlands planted with other species. Plant growth and fine root （root diameter ≤ 3 mm） biomass were related to removal efficiency. Fine root biomass rather than the mass of the entire root system played an important role in wastewater treatment. Removal efficiency varied with season and plant growth. Wetlands vegetated by P. purpureum significantly outperformed wetlands with other plants in May and June, whereas wetlands vegetated by P. communis and C. indica demonstrated higher removal efficiency from August to December. These findings suggest that abundance of fine roots is an important factor to consider in selecting for highly effective wetland plants. It also suggested that a plant community consisting of multiple plant species with different seasonal growth patterns and root characteristics may be able to enhance wetland performance.     

Characterisation of <Emphasis Type="Italic">Solanum lycopersicoides</Emphasis> Dun. root primordia culture with plant recovery

A liquid meristematic root primordia culture (RPC) of Solanum lycopersicoides Dun. based on persistent rhizogenesis in a modified Murashige and Skoog (1962) medium supplemented with NAA (15 mg·l⁻¹) or 2,4-D (1 mg·l⁻¹) was described. The meristematic clumps (2–3 mm in diameter) originating from NAA supplemented medium were capable of regenerating plants through the callus stage (up to 70 %). Efficient direct plant regeneration (up to 21 %) was possible from numerous single globular-shaped root primordia (RP) structures liberated from the parental aggregates in 2,4-D supplemented proliferation medium without NH₄NO₃ and with a 2.5 fold increase in KNO₃. The RP converted into plantlets (artificial seedlings) on solid or liquid media without growth growth regulators through the unipolar followed by the mace-shaped bipolar structure stages. The use of apical shoot bud, root apices or root segments as a primary explants brought about RPC induction and plant regeneration. The plants derived from 2 years old culture were phenotypically identical to their parental S. lycopersicoides plants and possessed the same ploidy.     

Development of a transgenic hairy root system in jute (Corchorus capsularis L.) with gusA reporter gene through Agrobacterium rhizogenes mediated co-transformation

Transgenic hairy root system is important in several recalcitrant plants, where Agrobacterium tumefaciens-mediated plant transformation and generation of transgenic plants are problematic. Jute (Corchorus spp.), the major fibre crop in Indian subcontinent, is one of those recalcitrant plants where in vitro tissue culture has provided a little success, and hence, Agrobacterium-mediated genetic transformation remains to be a challenging proposition in this crop. In the present work, a system of transgenic hairy roots in Corchorus capsularis L. has been developed through genetic transformation by Agrobacterium rhizogenes harbouring two plasmids, i.e. the natural Ri plasmid and a recombinant binary vector derived from the disarmed Ti plasmid of A. tumefaciens. Our findings indicate that the system is relatively easy to establish and reproducible. Molecular analysis of the independent lines of transgenic hairy roots revealed the transfer of relevant transgenes from both the T-DNA parts into the plant genome, indicating the co-transformation nature of the event. High level expression and activity of the gusA reporter gene advocate that the transgenic hairy root system, thus developed, could be applicable as gene expression system in general and for root functional genomics in particular. Furthermore, these transgenic hairy roots can be used in future as explants for plantlet regeneration to obtain stable transgenic jute plants.     

Effects of Tiller Separation and Root Pruning on the Growth of Lolium perenne L.

Experiments have been carried out to assess the role of inter-tillerinteractions within plants of Lolium perenne L. cv. S24 as factorswhich could play a part in determining the growth-rate of thewhole plant. In order to do this, the effect of tiller separationon plant growth was studied as well as the influence of theremoval of different parts of the root and shoot system. Wheneverplants were supplied with nutrients by means of culture solutions,separation of tillers brought about a marked increase in theamount of dry matter produced. Root pruning and in particularthe removal of root apices brought about similar increases ingrowth. Separation of tillers in soil-grown plants did not bringabout increased growth. These results are interpreted as indicatingthat separation of tillers or the initial removal of root apicesin plants supplied with nutrients by means of culture solution,promotes root branching which brings about an increased netassimilation rate arising from an increase supply of a growth-promotingsubstance, probably a cytokinin, from the root apices. The resultsare not taken as indicative of an intertiller interaction whichrestricts the growth of intact plants.     

The use of aeroponics to investigate antioxidant activity in the roots of <Emphasis Type="Italic">Xerophyta viscosa</Emphasis>

In order to ultimately understand the whole plant mechanism of attaining desiccation tolerance, we undertook to investigate the root tissues of the resurrection plant Xerophyta viscosa, as previous work has only been conducted on the leaf tissues of resurrection plants. An aeroponic plant growth system was designed and optimised to observe the root’s response to desiccation without the restrictions of a soil medium, allowing easy access to roots. Successful culture of both X.viscosa and the control, Zea mays, was achieved and dehydration stress was implemented through reduction of nutrient solution spraying of the roots. After drying to the air dry state (achieved after 7 days for roots and 10 days for shoots), rehydration was achieved by resumption of root spraying. X.viscosa plants survived desiccation and recovered but Z. mays did not. The activity of the antioxidant enzymes superoxide dismutase, catalase, ascorbate peroxidase and glutathione reductase and quantities of ascorbate and glutathione were determined during root desiccation. There was an initial decline in activity in all enzymes upon drying to 80% RWC, but activity thereafter remained constant, at rates indicative of potential metabolic activity, to the air-dry state. This data suggests that these enzymes are not denatured by desiccation of the root tissue. Ascorbate and glutathione content remained constant at concentrations of 70 and 100 μM, respectively during drying. Thus root tissues appear to retain antioxidant potential during drying, for use in recovery upon rehydration, as has been reported for leaf tissues of this and other resurrection plants.     

Siderophore mediated plant growth promotion at low temperature by mutant of fluorescent pseudomonad⋆

A cold resistant mutant of Pseudomonas fluorescens ATCC 13525 was developed, which could grow equally well at 25 and 10 °C and its effect on plant growth promotion under in vitro and in situ conditions was observed. Siderophore estimation revealed it to be a siderophore-overproducing mutant (17-fold increase) when compared to its wild type counterpart. A gnotobiotic root elongation assay indicated that the mutant (CRPF₉) promoted growth more than its wild type both at 25 and 10 °C, indicating its effectiveness at low temperature. Further, root colonization studies showed that CRPF₉ was an efficient rhizosphere colonizer, inducing a significant increase in root (35%) and shoot length (28%) of mung bean plants in unsterilized soil system. The persistence and stability of the mutant was evident in rhizospheric soil. A sand culture experiment showed that ferric citrate was better than Fe(OH)₃ as an iron source for plant growth, but in the presence of CRPF₉ both salts were comparable. This study demonstrates the potential of chemical mutagenesis for improving the plant growth promoting properties of a P. fluorescens strain and its stimulating impact on plant growth promotion at low temperature both under in vitro and in situ conditions.