Direct Regeneration of Shoots from Hairy Root Cultures of Centaurium erythraea Inoculated with Agrobacterium rhizogenes (original) (raw)
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Plant Cell Reports, 2006
Hairy roots of Centaurium erythraea were obtained by infection with Agrobacterium rhizogenes strain LBA 9402. They spontaneously regenerated adventitious shoots in Woody Plant liquid medium without growth regulators. The shoots were grown continuously in Murashige and Skoog (MS) liquid or agar solidified media supplemented with 0.1 mg l −1 indole-3-acetic acid and 1.0 mg l −1 6-benzylaminopurine. These shoots produced roots 4 weeks after transfer into agar-solidified MS medium without phytohormones. Regenerated plants grown and flowered under greenhouse conditions. The transgenic value of the regenerated plants was confirmed by the polymerase chain reaction amplification. Transformation by Agrobacterium rhizogenes alters plant morphology and production of secoiridoid glucosides. The level of secoiridoids was also modified by development stage of transformed plants. The total content of the compounds (expressed as the sum of gentiopicroside, sweroside and swertiamarin) in 10-week old pRi-transformed regenerants was 280 mg g −1 dry weight and was 8-times the content in the sample of commercially available C. erythraea herb.
Agrobacterium-mediated transformation of the medicinal plant Centaurea montana
Plant Cell, Tissue and …, 2011
An efficient transformation system was developed for Centaurea montana by co-cultivation of leaf explants with Agrobacterium tumefaciens strain AGL1 that contained a plasmid harboring the isopentenyl transferase gene under the control of the developmentally regulated Atmyb32 promoter of Arabidopsis thaliana and the gene encoding for hygromycin resistance under the control of the Cauliflower Mosaic Virus 35S (CaMV35S) promoter. A total of 990 explants were infected with Agrobacterium, and 18 shoots were regenerated resulting in an overall transformation efficiency of 1.8%. Molecular analyses, including PCR, Southern blotting and RT-PCR, were performed on T 0 and T 1 plants to confirm chromosomal integration and expression of the transgene in the phenotypically normal transformed plants. Transformation of C. montana was also performed using A. tumefaciens supervirulent strain EHA105 harboring the b-glucuronidase (GUS) reporter gene. Expression of the GUS gene in the putative transgenics was confirmed using a histochemical GUS assay.
Plant Cell Reports, 2000
Hairy roots were induced from leaf segments of Crotalaria juncea, which is used as a green manure crop antagonistic to nematodes, by infection with a mikimopine type wild strain of Agrobacterium rhizogenes A13 (MAFF02-10266). These roots exhibited vigorous growth and abundant lateral branching on half-strength Murashige and Skoog (1/2MS) medium without phytohormones. The adventitious shoots were induced from 30% of root segments 3 months after transfer onto medium containing 3 mg/l benzyl adenine. These shoots produced roots 1 month after transfer onto 1.2% agar-solidified 1/2MS medium without phytohormones. Regenerated plants were successfully grown under greenhouse conditions. The transgenic nature of the regenerated plants was confirmed by Southern-blot analysis.
Hairy Roots: An Ideal Platform for Transgenic Plant Production and Other Promising Applications
Phytochemicals, Plant Growth, and the Environment, 2012
The infection of plants by Agrobacterium rhizogenes results in a "hairy root" phenotype characterized by rapid growth in hormone-free medium, an unusual ageotropism and extensive lateral branching. The pathological rhizogenicity of A. rhizogenes arises from the stable insertion of a region of the A. rhizogenes Ri (root-inducing) plasmid into the plant nuclear genome. This plasmid can be engineered to contain foreign genes, which can also be stably inserted into the host genome. As such, A. rhizogenes represents a viable alternative for the genetic transformation of plant tissue not readily transformed by A. tumefaciens . However, to be effective as a genetic transformation system, the routine regeneration of full plants from hairy root cultures is essential. In this chapter, we report on some important features of hairy roots, describe recent progress in the regeneration of plants from A. rhizogenes -derived hairy roots and provide a summary of selected applications. These include the use of A. rhizogenes as an ef fi cient system to boost rhizogenesis in recalcitrant plant species and to create new plant varieties and the use of hairy root cultures and A. rhizogenes -engineered plants for secondary metabolite production, in phytoremediation and for the production of recombinant proteins (i.e. molecular farming) for the healthcare industry.
The Agrobacterium rhizogenes pRi TL-DNA segment as a gene vector system for transformation of plants
MGG Molecular & General Genetics, 1987
A plant gene transfer system was developed from the Agrobacterium rhizogenes pRi15834 TL-DNA region. "Intermediate integration vectors" constructed from ColEl-derived plasmids served as cloning vectors in Escherichia coli and formed cointegrates into the TL-DNA after transfer to A. rhizogenes. An A. rhizogenes strain with pBR322 plasmid sequences replacing part of the TL-DNA was also constructed. Plasmids unable to replicate in Agrobacterium can integrate into this TL-DNA by homologous recombination through pBR322 sequences. No loss of pathogenicity was observed with the strains formed after integration of intermediate vectors or strains carrying pBR322 in the TL-DNA segment. Up to 15 kb of DNA have been transferred to plant cells with these systems. The T-DNA from a binary vector was cotransformed into hairy roots which developed after transfer of the wild-type pRi T-DNA. Tested on Lotus corniculatus the TL-derived vector system transformed 90% of the developed roots and the T-DNA from the binary vector was cotransformed into 60% of the roots. Minimum copy numbers of one to five were found. Both constitutive and organ-specific plant genes were faithfully expressed after transfer to the legume L. eorniculatus.
2009
In vitro raised shoots of Plumbago rosea L. were infected with A4 strain of Agrobacterium rhizogenes to initiate hairy root formation, which produced 3.0±0.33 hairy roots per incision on explants in 20 d incubation. Southern blot analysis confirmed the integration of T-DNA into the genome of the roots. The hairy roots were cultured on MS agar medium supplemented with 2.0 mg/L BAP to induce the formation of shoots (3.2±0.24) of 0.2-0.4 cm length in 7-8 wks. Isolated shoots were multiplied through sub culturing in the presence of 0.5 mg/L BAP and the resultant shoots were subjected to combined elongation (3.29±0.16 cm) and rooting (12.6±0.57) in a medium supplemented with 0.1 mg/L IBA. The rooted plants were invariably abnormal with short internodes and wrinkled leaves showing 34.5% establishment in 2 months after transplantation in polybags and rearing under 75% sunlight in a shade house. Out of 38 plants transferred to the field, 20 (52.6%) survived and grew over 10-month period to ...
Plant Cell Reports, 2004
Agrobacterium rhizogenes strain LBA9402 was used to transform Pinus halepensis embryos, seedlings and shoots. Mature embryos exhibited susceptibility to the agrobacterium as monitored by ~-ghicuronidase (GUS) expression, with more than 85% showing considerable transient GUS expression in the radicle. GUS expression was also observed in cotyledons, but at a lower rate of about 24% of the embryos (1-5 spots/embryo). Stable transformation was evidenced by the regeneration of GUS-expressing roots and calli from infected P. halepensis seedlings. Inoculum injections into intact seedling hypocotyls induced callus and root formation at the wound sites in 64% of the seedlings. Dipping seedling cuttings in a bacterial suspension resulted in adventitious root formation in 71% of the seedling cuttings, all of which expressed GUS activity. Adventitious shoots, that were induced on 2.5-year-old seedlings by pruning and spraying with 6benzylaminopurine, were infected by injecting of bacterial suspension into their basal side. Two months later, adventitious roots and root primordia regenerated in 74% and 40% of 2-and 5-month-old shoots, respectively. Non-transformed shoots, either without or with auxin application, failed to form roots. Polymerase chain reaction and Southern blot analyses confirmed the uidA-transgenic nature of the root and callus, as well as the presence of rolC and rolB genes in roots from infected P. halepensis seedlings.
Plant Biotechnology Reports, 2007
A protocol for induction and establishment of Agrobacterium rhizogenes-mediated hairy root cultures of Picrorhiza kurroa was developed through optimization of the explant type and the most suitable bacterial strain. The infection of leaf explants with the LBA9402 strain resulted in the emergence of hairy roots at 66.7% relative transformation frequency. Nine independent, opine and TL-positive hairy root clones were studied for their growth and specific glycoside (i.e., kutkoside and picroside I) productivities at different growth phases. Biosynthetic potentials for the commercially desirable active constituents have been expressed by all the tested hairy root clones, although distinct inter-clonal variations could be noted in terms of their quantity. The yield potentials of the 14-P clone, both in terms of biomass as well as individual glycoside contents (i.e., kutkoside and picroside I), superseded that of all other hairy root clones along with the non-transformed, in vitro-grown control roots of P. kurroa. The present communication reports the first successful establishment, maintenance, growth and selection of superior hairy root clone of Picrorhiza kurroa with desired phyto-molecule production potential, which can serve as an effective substitute to its roots and thereby prevent the indiscriminate up-rooting and exploitation of this commercially important, endangered medicinal plant species.