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1.
Medicago
truncatula, barrel medic, is a forage crop that has been developed into a model legume. The development of new transformation methods is important for functional genomic studies in this species. Based on Agrobacterium tumefaciens-mediated transformation of root explants, we developed an effective system for producing M. truncatula (genotype R108) transgenic plants. Among the four A. tumefaciens strains (AGL1, C58C1, EHA105 and LBA4404) tested, EHA105 and AGL1 were most effective in regenerating transgenics. Callus induction frequency from root explants was 69.8%, and plantlet/shoot regeneration frequency was 41.3% when EHA105 was used. Transgenic nature of the regenerated plants was confirmed by PCR and Southern hybridization analyses. Progeny analysis revealed stable Mendelian meiotic transmission of transgenes. Because M. truncatula is particularly useful for the study of root endosymbiotic associations, we further developed a plant regeneration system from A. rhizogenes-transformed hairy roots of M. truncatula. Fertile true transgenic plants were regenerated from the hairy roots, thus allowing the assessment of gene functions at the whole plant level. Segregation analysis revealed that the hairy root genes could be segregated out in the progenies. By coupling A. rhizogenes-mediated hairy root transformation and the regeneration system reported here, once potential genes of interest are identified, the transformed hairy roots carrying such genes could be directly regenerated into plants for more detailed characterization of the genes. 相似文献
2.
Key message
Both in vitro and in vivo hairy root transformation systems could not replace whole plant transformation for promoter analysis of root-specific and low-P induced genes in soybean.Abstract
An efficient genetic transformation system is crucial for promoter analysis in plants. Agrobacterium-mediated transformation is the most popular method to produce transgenic hairy roots or plants. In the present study, first, we compared the two different Agrobacterium rhizogenes-mediated hairy root transformation methods using either constitutive CaMV35S or the promoters of root-preferential genes, GmEXPB2 and GmPAP21, in soybean, and found the efficiency of in vitro hairy root transformation was significantly higher than that of in vivo transformation. We compared Agrobacterium rhizogenes-mediated hairy root and Agrobacterium tumefaciens-mediated whole plant transformation systems. The results showed that low-phosphorous (P) inducible GmEXPB2 and GmPAP21 promoters could not induce the increased expression of the GUS reporter gene under low P stress in both in vivo and in vitro transgenic hairy roots. Conversely, GUS activity of GmPAP21 promoter was significantly higher at low P than high P in whole plant transformation. Therefore, both in vitro and in vivo hairy root transformation systems could not replace whole plant transformation for promoter analysis of root-specific and low-P induced genes in soybean. 相似文献3.
BNYVV-derived dsRNA confers resistance to rhizomania disease of sugar beet as evidenced by a novel transgenic hairy root approach 总被引:1,自引:0,他引:1
Ourania I. Pavli Nicholas J. Panopoulos Rob Goldbach George N. Skaracis 《Transgenic research》2010,19(5):915-922
Agrobacterium rhizogenes-transformed sugar beet hairy roots, expressing dsRNA from the Beet necrotic yellow vein virus replicase gene, were used as a novel approach to assess the efficacy of three intron-hairpin constructs at conferring resistance
to rhizomania disease. Genetically engineered roots were similar in morphology to wild type roots but were characterized by
a profound abundancy, rapid growth rate and, in some cases, plagiotropic development. Upon challenge inoculation, seedlings
showed a considerable delay in symptom development compared to untransformed or vector-transformed seedlings, expressing dsRNA
from an unrelated source. The transgenic root system of almost all seedlings contained no or very low virus titer while the
non-transformed aerial parts of the same plants were found infected, leading to the conclusion that the hairy roots studied
were effectively protected against the virus. This readily applicable novel method forms a plausible approach to preliminarily
evaluate transgenic rhizomania resistance before proceeding in transformation and whole plant regeneration of sugar beet,
a tedious and time consuming process for such a recalcitrant crop species. 相似文献
4.
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
(KmR) 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 KmR 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 相似文献
5.
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. 相似文献
6.
Summary Inoculation of carrot discs and Lotus corniculatus plantlets with mixtures of different Agrobacterium rhizogenes or of A. rhizogenes and A. tumefaciens or with Agrobacterium strains harboring both an Ri and a modified Ti plasmid resulted in frequent multiple (pluribacterial) transformation of cells, as revealed by the mixed opine-type of hairy roots arising from them. Multiple transformation may account for the presence of dispersed T-DNA inserts in crown gall and hairy root lines. A plant genetic engineering strategy based on segregation of T-DNA inserts in the progeny of multiple transformants is proposed. 相似文献
7.
Cruciferous hairy roots are often used for improving drought adaptability, peroxidase production, andin vitro subculturing ofPlasmodiophora brassicae. For metabolic engineering,Agrobacterium tumefaciens-mediated systems have previously been developed for hairy root production in other plant species. Here, we used therolABC gene binary construct inA. tumefaciens strain GV3101 to establish cultures of Chinese cabbage hairy roots. On both solid and liquid media, therolABC hairy root lines exhibited a wild-type hairy root syndrome in terms of their growth and morphology. This demonstrates that
those three genes are sufficient to induce high-quality hairy roots in Chinese cabbage. Such a system could be useful for
the stable production of secondary metabolites in that species. 相似文献
8.
9.
Use of ri-mediated transformation for production of transgenic plants 总被引:12,自引:0,他引:12
Mary C. Christey 《In vitro cellular & developmental biology. Plant》2001,37(6):687-700
Summary
Agrobacterium rhizogenes-mediated transformation has been used to obtain transgenic plants in 89 different taxa, representing 79 species from 55 genera
and 27 families. A diverse range of dicotyledonous plant families is represented, including one Gymnosperm family. In addition
to the Ri plasmid, over half these plants have been transformed with foreign genes, including agronomically useful traits.
Plants regenerated from hairy roots often show altered plant morphology such as dwarfing, increased rooting, altered flowering,
wrinkled leaves and/or increased branching due to rol gene expression. These altered phenotypic features can have potential applications for plant improvement especially in the
horticultural industry where such morphological alterations may be desirable. Use of A. rhizogenes and rol gene transformation has tremendous potential for genetic manipulation of plants and has been of particular benefit for improvement
of ornamental and woody plants. 相似文献
10.
Dong Cao Wensheng Hou Wei Liu Weiwei Yao Cunxiang Wu Xiaobing Liu Tianfu Han 《Plant Cell, Tissue and Organ Culture》2011,107(3):541-552
Salinity is a major factor resulting in extensive loss of agricultural production. Genetic transformation has become a powerful
tool for studying gene function and for improving crop salt tolerance. In this study, a TaNHX2 gene was transformed into a plant cloning vector under the control of cauliflower mosaic virus 35S promoter, and then introduced
into Agrobacterium rhizogenes strain K599. Explants of soybean were transformed with A. rhizogenes and ‘composite’ plants consisting of wild-type shoots and transgenic hairy roots overexpressing TaNHX2 were produced. When exposed to salt stress, ‘composite’ plants displayed high salinity tolerance at 171 mM NaCl in vermiculite
and in solid medium supplemented with up to 200 mM NaCl, whereas control plants displayed chlorosis and died within 15 days
under above treatment conditions. We subsequently obtained soybean plants overexpressing TaNHX2 through A. tumefaciens-mediated transformation and studied four homozygous lines of TaNHX2. Transgenic lines displayed an enhanced salt tolerance in plant biomass and flower number per plant, compared with wild type
plants grown on sand culture containing 150 mM NaCl. Furthermore, transgenic plants of line C12-11 showed longer survival,
less growth inhibition and greater number of flowers than wild type plants. Taken together, these results indicated that TaNHX2 gene could enhance salt tolerance of soybean, and A. rhizogenes-mediated transformation system could be used as a complementary tool of A. tumerfaciens-mediated transformation to rapidly investigate candidate gene function in soybean. 相似文献
11.
Nadiia Matvieieva Volodymyr Duplij Yakiv Ratushniak Anatolij Shakhovsky Tetiana Kyrpa-Nesmiian 《Preparative biochemistry & biotechnology》2019,49(1):82-87
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. 相似文献
12.
13.
Puddephat I.J. Robinson H.T. Fenning T.M. Barbara D.J. Morton A. Pink D.A.C. 《Molecular breeding : new strategies in plant improvement》2001,7(3):229-242
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 TL 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 TL-DNA and vector T-DNA. GUS-positive phenotypically normal progeny free of TL-DNA were identified in three transgenic lines out of the six tested representing all the cultivars regenerated including both cauliflower and broccoli. 相似文献
14.
Bushra Hafeez Kiani John Suberu Guy Cameron Barker Bushra Mirza 《In vitro cellular & developmental biology. Plant》2014,50(5):590-600
Extensive studies have been carried out for the optimization of regeneration and transformation conditions for both Agrobacterium tumefaciens- and Agrobacterium rhizogenes-mediated transformation of the highly medicinal plant Artemisia annua. Most protocols describe laborious transformation procedures requiring no less than 3 mo to obtain transgenic plants. This study reports rapid and efficient protocols for A. tumefaciens- and A. rhizogenes-mediated transformation of A. annua, which were equally effective for transformation of Artemisia dubia. In both transformation procedures, stem explants responded best for maximal production of transformed plants and hairy roots. In the case of A. tumefaciens-mediated transformation, stem explants were pre-cultured for 2 d followed by infection with A. tumefaciens strain LBA4404 for 48 h. A. annua explants showed maximal transformation rate (43.5%) on half-strength Murashige and Skoog medium containing 40 mg/L kanamycin in only 20 d. The same method was tested using a related species A. dubia and resulted in a transformation rate of 41.3%, demonstrating that this protocol is efficient and genotype-independent. In the case of A. rhizogenes-mediated transformation for the production of hairy root cultures, in vitro-grown stem explants were infected with a single colony of A. rhizogenes strain LBA9402 by creating incisions at different places of the stem explants, which resulted in production of hairy roots in only 7 d. The method was tested in both A. annua and A. dubia, which resulted in transformation rates of 90 and 87.5%, respectively. Integration of the transgene and copy number was confirmed by PCR and Southern blot analyses, respectively. The miniprep transformation protocols developed for both A. tumefaciens- and A. rhizogenes-mediated transformation are simple, efficient, and potentially applicable to other species of Artemisia for transfer of pharmaceutically important genes. 相似文献
15.
To establish a procedure for Agrobacterium tumefaciens-mediated transformation of golden pothos (Epipremnum aureum) plants, the effects of selection antibiotics and the preculture period of stem explants before A. tumefaciens infection were examined. Explants were co-cultivated with A. tumefaciens EHA105, harboring the plasmid pGWB2/cGUS, on a somatic embryo-inducing medium supplemented with acetosyringone. Resulting
transgenic somatic embryos were screened on an antibiotic selection medium, and the transgenic pothos plants were regenerated
on a germination medium. Hygromycin was the optimum selection antibiotic tested. The preculture period significantly affected
the transformation efficiency, with explants precultured for one-day showing the best efficiency (5–30%). Both transformed
hygromycin-resistant embryos and regenerated plants showed β-glucuronidase activity. Southern blot analysis confirmed transgene
integration into the pothos genome. This reproducible transformation system for golden pothos may enable the molecular breeding
of this very common indoor plant. 相似文献
16.
Ye X Williams EJ Shen J Johnson S Lowe B Radke S Strickland S Esser JA Petersen MW Gilbertson LA 《Transgenic research》2011,20(4):773-786
Single transgene copy, vector backbone-free transgenic crop plants are highly desired for functional genomics and many biotechnological
applications. We demonstrate that binary vectors that use a replication origin derived from the Ri plasmid of Agrobacterium rhizogenes (oriRi) increase the frequency of single copy, backbone-free transgenic plants in Agrobacterium tumefaciens mediated transformation of soybean, canola, and corn, compared to RK2-derived binary vectors (RK2 oriV). In large scale soybean
transformation experiments, the frequency of single copy, backbone-free transgenic plants was nearly doubled in two versions
of the oriRi vectors compared to the RK2 oriV control vector. In canola transformation experiments, the oriRi vector produced
more single copy, backbone-free transgenic plants than did the RK2 oriV vector. In corn transformation experiments, the frequency
of single copy backbone-free transgenic plants was also significantly increased when using the oriRi vector, although the
transformation frequency dropped. These results, derived from transformation experiments using three crops, indicate the advantage
of oriRi vectors over RK2 oriV binary vectors for the production of single copy, backbone-free transgenic plants using Agrobacterium-mediated transformation. 相似文献
17.
Hairy Root-activation Tagging: a High-throughput System for Activation Tagging in Transformed Hairy Roots 总被引:2,自引:0,他引:2
Activation tagging is a powerful technique for generating gain-of-function mutants in plants. We developed a new vector system
for activation tagging of genes in “transformed hairy roots”. The binary vector pHR-AT (Hairy Root-Activation Tagging) and
its derivative pHR-AT-GFP contain a cluster of rol (rooting locus) genes together with the right border facing four tandem repeats of the cauliflower mosaic virus (CaMV) 35S
enhancer element on the same T-DNA. Transformation experiments using Arabidopsis, potato, and tobacco as model plants revealed that upon inoculating plants with Agrobacterium tumefaciens harboring these vectors, a large number of independently transformed roots could be induced from explants within a short
period of time, and root culture lines were subsequently established. Molecular analyses of the pHR-AT-GFP-transformed Arabidopsis lines showed that expression of the genes adjacent to the T-DNA insertion site was significantly increased. This system may
facilitate application of the activation-tagging approach to plant species that are recalcitrant to the regeneration of transgenic
plants. High-throughput metabolic profiling of activation-tagged root culture lines will offer opportunities for identifying
regulatory or biosynthetic genes for the production of valuable secondary metabolites of interest. 相似文献
18.
Segregation of genes transferred to one plant cell from two separate Agrobacterium strains 总被引:22,自引:0,他引:22
Agrobacterium tumefaciens and Agrobacterium rhizogenes are soil bacteria which transfer DNA (T-DNA) to plant cells. Two Agrobacterium strains, each with a different T-DNA, can infect plants and give rise to transformed tissue which has markers from both T-DNAs. Although marker genes from both T-DNAs are in the tissue, definitive proof that the tissue is a cellular clone and that both T-DNAs are in a single cell is necessary to demonstrate cotransformation. We have transferred two distinguishable T-DNAs, carried on binary vectors in separate Agrobacterium rhizogenes strains, into tomato cells and have recovered hairy roots which received both T-DNAs. Continued expression of marker genes from each T-DNA in hairy roots propagated from individual root tips indicated that both T-DNAs were present in a single meristem. Also, we have transferred the two different T-DNAs, carried on identical binary vector plasmids in separate Agrobacterium tumefaciens strains, into tobacco cells and recovered plants which received both T-DNAs. Transformed plants with marker genes from each T-DNA were outcrossed to wild-type tobacco plants. Distribution of the markers in the F1 generation from three cotransformed plants of independent origin showed that both T-DNAs in the plants must have been present in the same cell and that the T-DNAs were genetically unlinked. Cotransformation of plant cells with T-DNAs from two bacterial strains and subsequent segregation of the transferred genes should be useful for altering the genetic content of higher plants. 相似文献
19.
Y. Q. Zhou H. Y. Duan C. E. Zhou J. J. Li F. P. Gu F. Wang Z. Y. Zhang Z. M. Gao 《Russian Journal of Plant Physiology》2009,56(2):224-231
The objective of this research was to establish an efficient system of genetic transformation and plant regeneration from
hairy roots by infecting the leaf sections and stem segments of in vitro Rehmannia glutinosa Libosch. f. hueichingensis Hsiao plantlets. Hairy roots were induced from them after co-culturing with Agrobacterium rhizogenes strain 15834 at a frequency of 32 and 29.4%, respectively. The calluses were induced from hairy roots on half-strength Murashige
and Skoog medium containing 0.2 mg/l kinetin and 3.0 mg/l benzyladenine at a frequency of 100%, from which transgenic shoots
and plantlets were developed. Transgenic plantlets did not have differences in morphology except the shortened internodes
and an increase in adventitious root formation compared to wild-type plants. PCR and Southern-blot analyses confirmed that
rolB gene of TL-DNA was inserted in the genome of transformed hairy roots and plantlets. RT-PCR analysis and opine paper electrophoresis
revealed that rolB gene was expressed in the transformed hairy roots and plantlets. Conclusively, transgenic hairy roots and transgenic plants
of Rehmannia glutinosa Libosch. f. hueichingensis Hsiao were developed for the first time.
This text was submitted by the authors in English.
Published in Russian in Fiziologiya Rastenii, 2009, Vol. 56, No. 2, pp. 247–255. 相似文献