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1.
Genetic transformation of plants mediated by Ri plasmid of Agrobacterium rhizogenes occupies a special place in plant cell engineering, since this technique based on a natural phenomenon allows cultivation of isolated growing plant roots on hormone-free media. Application of wild-type unmodified agrobacterial strains allows us to obtain root cultures capable of long-term growth in vitro due to an increased sensitivity of the cells to auxins while other biochemical properties remain unaltered. A collection of pRi T-DNA transformed roots of certain dicotyledons was made; some strains in it are used to study synthesis of secondary metabolites in root cells. The in vitro cultivated roots could synthesize root-specific metabolites, which makes possible their application for large-scale biotechnological production of ecologically pure crude drugs. Cocultivation of pRi T-DNA transformed roots with arbuscular mycorrhizal fungi makes possible vital study of all stages of obligate symbiont development and interaction with plant roots. Dual axenic culture of AM fungi and pRi T-DNA transformed plants can be used to make a collection of the most valuable endomycorrhizal fungal species and to produce considerable quantities of homogeneous fungal inoculums. 相似文献
2.
To test whether plant growth–promoting bacteria might be useful in facilitating the growth of Phragmites australis, the common reed, in the presence of metals and organic compounds, P. australis seeds were treated with plant growth–promoting bacteria. The bacterium Pseudomonas asplenii AC was genetically transformed to express a bacterial gene encoding the enzyme 1-aminocyclopropane-1-carboxylate deaminase,
and both the native and transformed bacteria were tested in conjunction with P. australis. Inoculation of seeds, which were subsequently grown in the presence of copper or creosote, with transformed P. asplenii AC significantly increased seed germination. Moreover, the addition of either native or transformed P. asplenii AC to P. australis seeds enabled the plants (shoots and roots) to attain a greater size than noninoculated plants after growth in soil in the
presence of either copper or creosote. 相似文献
3.
The technique for the refinement of pRi T-DNA-transformed root cultivation by the root fragment encapsulation in the gel coat,
i.e., so-called “artificial seed” (AS) production, was studied. AS were produced from genetically transformed roots of Baikal
skullcap ( Scutellaria baicalensis Georgi) and common rue ( Ruta graveolens L.). The effects of duration of AS storage at 4°C on their subsequent growth activity and a capability for resumption of
actively growing root cultures were analyzed. Encapsulation of contaminated Baikal skullcap root culture with the addition
of antibiotic and storage during 2–5 weeks at low above-zero temperature resulted in a complete elimination of infection,
i.e., obtaining the healthy root culture. Growth activity and total flavone concentration were markedly increased in this
culture, so that total productivity of this renewed root culture increased substantially. Using AS produced from the root
fragments of common rue, it was shown that, after long-term storage at low above-zero temperature, they are capable of not
only root growth resumption but also active shoot formation, which is of interest for plant micropropagation. Long-term retaining
growth activity of AS produced from root cultures of valuable medicinal plants permits their usage as a reserve and also,
in the case of necessity, for long-distance transport as compact axenic root inocula. The storage of viable root fragments
within AS also helps to optimize intervals between numerous subculturings of root cultures required for the maintenance of
IPPRAS collection in the active state. 相似文献
4.
The composition of essential oil obtained by hydrodistillation from genetically transformed roots of common rue ( Ruta graveolens L.) was analyzed. Using gas chromatography and complex gas chromatography-mass spectrometry, it was established that the
major component of rue essential oil was a root-specific sesquiterpene geijerene comprising 67% of total amount of volatile
compounds. In essential oil of cultured rue roots, furocoumarins characteristic of intact plant roots were found, viz. osthole,
halepensin, and rutacultin. The content of essential oil in genetically transformed rue roots was 0.23% of root dry weight,
which is comparable with that in the roots of intact plants. The long-term maintenance in the in vitro cultured rue roots
of a capability for the synthesis of essential oil major components characteristic of intact plants allows their usage for
studying the physiological activity of these volatile compounds and their putative role in the plant root interaction in biocenoses. 相似文献
5.
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. 相似文献
6.
Agrobacterium rhizogenes causes hairy root disease in plants. The neoplastic roots produced by A. rhizogenes infection is characterized by high growth rate and genetic stability. These genetically transformed root cultures can produce higher levels of secondary metabolites or amounts comparable to that of intact plants. Hairy root cultures offer promise for production of valuable secondary metabolites in many plants. The main constraint for commercial exploitation of hairy root cultures is their scaling up, as there is a need for developing a specially designed bioreactor that permits the growth of interconnected tissues unevenly distributed throughout the vessel. Rheological characteristics of heterogeneous system should also be taken into consideration during mass scale culturing of hairy roots. Development of bioreactor models for hairy root cultures is still a recent phenomenon. It is also necessary to develop computer-aided models for different parameters such as oxygen consumption and excretion of product to the medium. Further, transformed roots are able to regenerate genetically stable plants as transgenics or clones. This property of rapid growth and high plantlet regeneration frequency allows clonal propagation of elite plants. In addition, the altered phenotype of hairy root regenerants (hairy root syndrome) is useful in plant breeding programs with plants of ornamental interest. In vitro transformation and regeneration from hairy roots facilitates application of biotechnology to tree species. The ability to manipulate trees at a cellular and molecular level shows great potential for clonal propagation and genetic improvement. Transgenic root system offers tremendous potential for introducing additional genes along with the Ri T-DNA genes for alteration of metabolic pathways and production of useful metabolites or compounds of interest. This article discusses various applications and perspectives of hairy root cultures and the recent progress achieved with respect to transformation of plants using A. rhizogenes. 相似文献
7.
Hairy roots of Rhamnus fallax Boiss. were induced using Agrobacterium rhizogenes strain A4M70GUS. The culture established on Woody plant media (WPM) showed a typical hairy root phenotype: rapid growth,
reduced apical dominance and root plagiotropism. Seven clones of R. fallax were selected on the basis of their differences in colour and the root branching. The growth of hairy root culture, measured
through gain in fresh mass, was done under 16-h photoperiod or in the dark. An increase in anthraquinone (AQ) content was
obtained in clones with yellow and less branched roots, like clone 1 [16.43 mg g −1(d.m.)] and clone 7 [14.21 mg g −1(d.m.)], compared with other analysed transformed and non-transformed tissue. This study presents the first report of successful
transformation of any species from family Rhamnaceae by A. rhizogenes and analysis of AQ production in transformed tissue. 相似文献
8.
We examined the effects of genetic transformation by Agrobacterium rhizogenes on the production of tylophorine, a phenanthroindolizidine alkaloid, in the Indian medicinal plant, Tylophora indica. Transformed roots induced by the bacterium grew in axenic culture and produced shoots or embryogenic calli in the absence of hormone treatments. However, hormonal treatment was required to regenerate shoots in root explants of wild type control plants. Transformed plants showed morphological features typically seen in transgenic plants produced by A. rhizogenes, which include, short internodes, small and wrinkled leaves, more branches and numerous plagiotropic roots. Plants regenerated from transformed roots showed increased biomass accumulation (350–510% in the roots and 200–320% in the whole plants) and augmented tylophorine content (20–60%) in the shoots, resulting in a 160–280% increase in tylophorine production in different clones grown in vitro. 相似文献
9.
Small callus pieces excised from the Agrobacterium transformed root line D 2 of Datura stramonium, were cultured onto solidified MS medium supplemented with a 1.0 μM kinetin and three different concentrations (0.1, 0.5
and 1.0 μM) of 2,4-dichlorophenoxyacetic acid (2,4-D), and were examined for their alkaloid productivity in relation to organization
level and growth rate. Growth of transformed roots (in a MS liquid medium without plant growth regulators) was greater than
that of transformed calli excised from them and cultured separately. The addition of 1.0 μM 2,4-D to the culture medium had
a positive effect on callus biomass production, while it inhibited root formation by this tissue (the lower the 2,4-D concentration
in the medium the greater the number of roots which emerged from the calli). Hyoscyamine production was also higher in the
transformed roots than in the transformed calli, and in these tissues the production of hyoscyamine was positively correlated
with organogenesis index ( i.e. its ability for rooting). At the same time, the epoxidation of hyoscyamine to scopolamine only took place in the transformed
calli. This occurred to a greater extent at the lower concentrations of 2,4-D in the culture medium. The mode through which
the 2,4-D could control the alkaloid production of transformed callus is discussed. 相似文献
10.
In this study, morphological alterations, biomass growth, and secondary metabolite production of genetically transformed hairy
roots of Panax ginseng C. A. Meyer, were evaluated after administration of plant growth regulators. The addition of benzylamino purine and kinetin
to the culture media increased biomass formation and phenolic compound biosynthesis in the hairy roots. α-Naphthaleneacetic
acid and indole-3-butyric acid inhibited hairy root growth, however, low concentrations of indole-3-acetic acid slightly increased
hairy root growth. Low concentrations of 2,4-Dichlorophenoxyacetic acid profoundly inhibited growth of hairy roots. The addition
of plant growth regulators, such as auxin, did not increase total phenolic compounds in hairy roots that did not contain gibberellic
acid and cytokinins. Callus formation was induced in cultures suspended in liquid medium amended with benzylamino purine and
kinetin. Hairy roots regenerated from these calluses exhibited an active growth pattern with extensive lateral branching in
non-amended medium, similar to the growth pattern of the original hairy roots. 相似文献
11.
The possibility of rapid validation and functional analysis of nematode resistance genes is a common objective for numerous species and particularly for woody species. In this aim, we developed an Agrobacterium rhizogenes-mediated transformation protocol for Coffea arabica enabling efficient and rapid regeneration of transformed roots from the hypocotyls of germinated zygotic embryos, and the subsequent production of composite plants. The A. rhizogenes strain A4RS proved to be the most virulent. High transformation efficiencies (70%) were obtained using a 2-week co-cultivation period at a temperature of 15–18°C. Using a p35S- gusA- int construct inserted in the pBIN19 binary plasmid, we could estimate that 35% of transformed roots were GUS positive (co-transformed). Using the GUS assay as visual marker, 40% composite plants bearing a branched co-transformed rootstock could be obtained after only 12 weeks without selection with herbicides or antibiotics. Transgenic coffee roots obtained with A. rhizogenes did not exhibit the ‘hairy’ disturbed phenotype and were morphologically similar to normal roots. PCR analyses demonstrated that all co-transformed roots were positive for the expected rolB and gusA genes. Transformed and non-transformed root systems from both susceptible and resistant varieties were inoculated with Meloidogyne exigua nematode individuals. Inoculation of composite plants from the Caturra susceptible variety resulted in the normal development of nematode larvae. Numbers of extracted nematodes demonstrated that transformed roots retain the resistance/sensibility phenotype of varieties from which they are derived. These results suggest that composite plants constitute a powerful tool for studying nematode resistance genes. 相似文献
12.
A steadily growing culture of genetically transformed roots of a valuable medicinal Altaic plant Hedysarum theinum Krasnob. was established using a Ri-plasmid (pRi) T-DNA of A4 wild strain of Agrobacterium rhizogenes. The composition of secondary substances accumulated in the in vitro roots and the roots of H. theinum intact seedlings was investigated. Isoflavonoids were found to represent by their main low-molecular metabolites. Preparative HPLC made it possible to isolate four substances from the methanolic extract of H. theinum cultured roots. Using 1H- and 13C-NMR-spectrometry, these substances were identified as formononetin, ononin (formononetin glycoside), malonyl ononin, and texasin glucoside. The qualitative composition of secondary metabolites of the genetically transformed roots and the roots of H. theinum seedlings was essentially the same, except that malonyl ononin was not found in the latter. The technique of producing artificial seeds on the basis of H. theinum roots cultured in vitro was tested, and the possibility of their use as a rhizogenic inoculum was substantiated. The culture of H. theinum roots is considered as a potential source of ecologically pure raw material for medicinal preparations, and the artificial seeds with root inoculum are a promising vehicle for propagation and conservation of this valuable plant. 相似文献
13.
ABSTRACTPlant cell cultivations are being considered as an alternative to agricultural processes for producing valuable phytochemicals. Since many of these products (secondary metabolites) are obtained by direct extraction from plants grown in natural habitat, several factors can alter their yield. The use of plant cell cultures has overcome several inconveniences for the production of these secondary metabolites. Organized cultures, and especially root cultures, can make a significant contribution in the production of secondary metabolites. Most of the research efforts that use differentiated cultures instead of cell suspension cultures have focused on transformed (hairy) roots. Agrobacterium rhizogenes causes hairy root disease in plants. The neoplastic (cancerous) roots produced by A. rhizogenes infection are characterized by high growth rate, genetic stability and growth in hormone free media. These genetically transformed root cultures can produce levels of secondary metabolites comparable to that of intact plants. Hairy root cultures offer promise for high production and productivity of valuable secondary metabolites (used as pharmaceuticals, pigments and flavors) in many plants. The main constraint for commercial exploitation of hairy root cultivations is the development and scaling up of appropriate reactor vessels (bioreactors) that permit the growth of interconnected tissues normally unevenly distributed throughout the vessel. Emphasis has focused on designing appropriate bioreactors suitable to culture the delicate and sensitive plant hairy roots. Recent reactors used for mass production of hairy roots can roughly be divided as liquid-phase, gas-phase, or hybrid reactors. The present review highlights the nature, applications, perspectives and scale up of hairy root cultures for the production of valuable secondary metabolites. 相似文献
14.
Agrobacterium rhizogenes induced hairy roots on discs of the tuber tissue of the tetraploid potato cv. Bintje after infection. Early and late generated hairy roots (transformed roots) were excised directly from the tuber discs and analysed for genetic stability by chromosome counts and determination of nuclear DNA content with flow cytometry. Another part was cloned by subculturing on MS-medium without hormones and subsequently analysed. Twenty-one of the 27 primary hairy roots and all 16 subcultured hairy roots were tetraploid thus suggesting genetic stability of transformed roots. Shoot regeneration was observed on hairy roots and therefore, it is suggested that A. rhizogenes transformation can be a suitable system for genetically stable plant regeneration from transformed cells. 相似文献
15.
Plant roots are known to orient growth through the soil by gravitropism, hydrotropism, and thigmotropism. Recent observations
of plant roots that developed in a microgravity environment in space suggested that plant roots may also orient their growth
toward oxygen (oxytropism). Using garden pea ( Pisum sativum L. cv. Weibul's Apollo) and an agravitropic mutant (cv. Ageotropum), root oxytropism was studied in the controlled environment
of a microrhizotron. A series of channels in the microrhizotron allowed establishment of an oxygen gradient of 0.8 mmol · mol −1 · mm −1. Curvature of seedling roots was determined prior to freezing the roots for subsequent spectrophotometric determinations
of alcohol dehydrogenase activity. Oxytropic curvature was observed all along the gradient in both cultivars of pea. The normal
gravitropic cultivar showed a maximal curvature of 45° after 48 h, while the agravitropic mutant curved to 90°. In each cultivar,
the amount of curvature declined as the oxygen concentration decreased, and was linearly related to the root elongation rate.
Since oxytropic curvature occurred in roots exposed to oxygen concentrations that were not low enough to induce the hypoxically
responsive protein alcohol dehydrogenase, we suspect that the oxygen sensor associated with oxytropism does not control the
induction of hypoxic metabolism. Our results indicate that oxygen can play a critical role in determining root orientation
as well as impacting root metabolic status. Oxytropism allows roots to avoid oxygen-deprived soil strata and may also be the
basis of an auto-avoidance mechanism, decreasing the competition between roots for water and nutrients as well as oxygen.
Received: 14 January 1998 / Accepted: 10 February 1998 相似文献
16.
We have explored cultures of roots transformed by Agrobacterium rhizogenes to test the availability of cadmium in sewage sludges. The toxic effects of Cd and the kinetics of Cd accumulation were examined for three species of transformed roots, grown for 2 weeks in nutrient media, containing Cd as a salt. Roots of sugar beet ( Beta vulgaris L.) were highly sensitive, while those of tobacco ( Nicotiana tobaccum L.) and morning glory ( Calystegia sepium R. Br) were more tolerant. Cd accumulation was higher in sugar beet and morning glory than in tobacco. We developed a non-sterile, 5-day procedure for testing the accumulation (an indication of availability) of Cd from sludge suspensions, using transformed roots of morning glory and tobacco. Cd accumulation varied with plant species and source of sludge. Ranking of Cd availability using this biological assay for Cd accumulation was confirmed by chemical tests with NH 4 acetate and EDTA. Results from transformed roots were also compared with those from normal, excised, tobacco roots and normal and transformed tobacco plantlets. No major alteration in Cd uptake was associated with genetic transformation. We thus demonstrated the feasibility of using transformed roots to estimate the availability of Cd in metal-contaminated materials like sewage sludges. 相似文献
17.
Hairy root cultures are often used to produce valuable metabolites. They are grown on sucrose-rich medium, which is highly susceptible to contamination. Trapa natans is a unique plant with photosynthesizing roots. It is a promising object to obtain photoautotrophic hairy root culture. Protocols for transformation of this species are yet unknown. We report that hairy roots can be induced in aquarium and in vitro cultures of T. natans by agrobacterium-mediated and biolistic transformation. 64 roots were induced by Agrobacterium rhizogenes strain 15834, two roots were obtained using strain K599. Strain A4 was not effective. Biolistics with either amplicons of rol genes and 1301 pCAMBIA plasmid carrying rol genes resulted in the formation of six roots. All these roots contained chloroplasts. This achievement opens a prospect for genetic transformation of T. natans and use of its green photosynthesizing hairy root cultures in production of bioactive substances and in phytoremediation. 相似文献
18.
A bioluminescence assay using recombinant Nitrosomonas europaea was adopted to detect and quantify natural nitrification inhibitors in plant–soil systems. The recombinant strain of N. europaea produces a distinct two-peak luminescence due to the expression of luxAB genes, introduced from Vibrio harveyi, during nitrification. The bioluminescence produced in this assay is highly correlated with NO 2− production ( r
2 = 0.94). Using the assay, we were able to detect significant amounts of a nitrification inhibitor produced by the roots of Brachiaria humidicola (Rendle) Schweick. We propose that the inhibitory activity produced/released from plants be termed ‘biological nitrification inhibition’ (BNI) to distinguish it from industrially produced inhibitors. The amount of BNI activity produced by roots was expressed in units defined in terms of the action of a standard inhibitor allylthiourea (AT). The inhibitory effect from 0.22 μM AT in an assay containing 18.9 mM of NH 4+ is defined as one AT unit of activity. A substantial amount of BNI activity was released from the roots of B. humidicola (15–25 AT unit g −1 root dry wt day −1). The BNI activity released was a function of the growth stage and N content of the plant. Shoot N levels were positively correlated with the release of BNI activity from roots ( r
2 = 0.76). The inhibitor/s released from B. humidicola roots suppressed soil nitrification. Additions of 20 units of BNI per gram of soil completely inhibited NO 3− formation in a 55-day study and remained functionally stable in the soil for 50 days. Both the ammonia monooxygenase and the hydroxylaminooxidoreductase enzymatic pathways in Nitrosomonas were effectively blocked by the BNI activity released from B. humidicola roots. The proposed bioluminescence assay can be used to characterize and determine the BNI activity of plant roots, thus it could become a powerful tool in genetically exploiting the BNI trait in crops and pastures. 相似文献
19.
Susceptibility of C. rubrum to Agrobacterium-mediated transformation was demonstrated by inoculating the petioles of in vitro grown plants with A. rhizogenes strain A4M70GUS. Hairy roots were produced in 8 % of explants. They were isolated and maintained on plant growth regulator-free
solid or liquid half-strength Murashige and Skoog medium for two years. Hairy root fresh mass increased 30 — 90 folds when
grown in liquid medium, which was superior to solid medium, where most of the hairy roots produced calli. When these calli
were grown on medium supplemented with 0.5 mg dm-3 thidiazuron, embryo-like structures were obtained. Transgenic status of
long-term callus and hairy root cultures was confirmed by histochemical GUS assay, by PCR specific to the uidA, rolA&B and ags genes and by Southern hybridization. 相似文献
20.
Plant species produce litter of varying quality and differ in the quality and quantity of compounds they release from live
roots, which both can induce different decomposer growth in the soil. To test whether differences in decomposer growth can
forecast the amount of N species acquire from plant litter, as suggested by theory, we grew individuals of three grassland
plants— Holcus lanatus, Plantago lanceolata and Lotus corniculatus—in soils into which 15N-labelled litter of either Holcus, Plantago or Lotus was added. We measured the effects of live roots and litter of each species on soil microbes and their protozoan and nematode
feeders, and to link decomposer growth and plant nutrient uptake, we measured the amount of N taken up by plants from the
added litter. We hypothesised that those species that induce the highest growth of microbes, and especially that of microbial
feeders, will also take up the highest amount of N from the litter. We found, however, that although numbers of bacterial-feeding
Protozoa and nematodes were on average lower after addition of Holcus than Plantago or Lotus litter, N uptake was higher from Holcus litter. Further, although the effects on Protozoa and bacterial- and fungal-feeding nematodes did not differ between the
live plants, litter-N uptake differed, with Holcus being the most efficient compared to Plantago and Lotus. Hence, although microbes and their feeders unquestionably control N mineralization in the soil, and their growth differs
among plant species, these differences cannot predict differences in litter-N uptake among plant species. A likely reason
is that for nutrient uptake, other species-specific plant traits, such as litter chemistry, root proliferation ability and
competitiveness for soil N, override in significance the species-specific ability of plants to induce decomposer growth. 相似文献
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