Electroporation Protocols for Agrobacterium

Main, Garry D.; Reynolds, S.; Gartland, Jill S.

doi:10.1385/0-89603-302-3:405

Cited by 21 publications

(21 citation statements)

References 5 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Competent cells of A. tumefaciens LBA4404, GV3101 and AGL1 strains were prepared according to Main et al (1995) and the binary vectors were introduced by electroporation. For the use of pGreen-derived vectors we cotransformed the A. tumefaciens cells with the pSoup vector (Hellens et al 2000a).…”

Section: Agrobacterium Tumefaciens Strainsmentioning

confidence: 99%

Agroinfiltration of grapevine leaves for fast transient assays of gene expression and for long-term production of stable transformed cells

et al. 2008

View full text Add to dashboard Cite

Agrobacterium-mediated transient assays for the analysis of gene function are used as alternatives to genetic complementation and stable plant transformation. Although such assays are routinely performed in several plant species, they have not yet been successfully applied to grapevines. We explored genetic background diversity of grapevine cultivars and performed agroinfiltration into in vitro cultured plants. By combining different genotypes and physiological conditions, we developed a protocol for efficient transient transformations of selected grapevine cultivars. Among the four cultivars analyzed, Sugraone and Aleatico exhibited high levels of transient transformation. Transient expression occurred in the majority of cells within the infiltrated tissue several days after agroinfiltration and, in a few cases, it later spread to a larger portion of the leaf. Three laboratory strains of Agrobacterium tumefaciens with different virulence levels were used for agroinfiltration assays on grapevine plants. This method promises to be a powerful tool to perform subcellular localization analyses. Grapevine leaf tissues were transformed with fluorescent markers targeted to cytoplasm (free GFP and mRFP1), endoplasmatic reticulum (GFP::HDEL), chloroplast (GAPA1::YFP) and mitochondria (b::GFP). Confocal microscope analyses demonstrated that these subcellular compartments could be easily visualized in grapevine leaf cells. In addition, from leaves of the Sugraone cultivar agroinfiltrated with endoplasmic reticulum-targeted GFP-construct, stable transformed cells were obtained that show the opportunity to convert a transiently transformed leaf tissue into a stably transformed cell line.

show abstract

Section: Agrobacterium Tumefaciens Strainsmentioning

confidence: 99%

Agroinfiltration of grapevine leaves for fast transient assays of gene expression and for long-term production of stable transformed cells

et al. 2008

View full text Add to dashboard Cite

show abstract

“…The SPDS1 coding sequence was amplified by PCR using the primers SPDS1F2 (5Ј-GTTCCCGGGATGGACGCTAAAGAAACC-3Ј) and SPDS1R2 (5Ј-GCCTCTAGAACGATCCTCCAGATTAGT-3Ј) and inserted as a SmaI-XbaI fragment into the expression cassettes of pPCV002-GIGI and pPCV821-MENCHU to label SPDS1 with C-terminal c-Myc and HA epitopes. The binary vectors were introduced into Agrobacterium strain GV3101 (pMP90RK) by electroporation (Main et al, 1995) and used in cotransformation experiments to express pairwise combinations of SPDS-myc and SPDS-HA proteins in cultured Arabidopsis cells, as described previously .…”

Section: Double Epitope Labeling and Expression Of Spds And Spms Protmentioning

confidence: 99%

A Polyamine Metabolon Involving Aminopropyl Transferase Complexes in Arabidopsis

Panicot

Minguet

Ferrando³

et al. 2002

Plant Cell

164

126

View full text Add to dashboard Cite

The conversion of putrescine to spermidine in the biosynthetic pathway of plant polyamines is catalyzed by two closely related spermidine synthases, SPDS1 and SPDS2, in Arabidopsis. In the yeast two-hybrid system, SPDS2 was found to interact with SPDS1 and a novel protein, SPMS (spermine synthase), which is homologous with SPDS2 and SPDS1. SPMS interacts with both SPDS1 and SPDS2 in yeast and in vitro. Unlike SPDS1 and SPDS2, SPMS failed to suppress the spe ⌬ 3 deficiency of spermidine synthase in yeast. However, SPMS was able to complement the spe ⌬ 4 spermine deficiency in yeast, indicating that SPMS is a novel spermine synthase. The SPDS and SPMS proteins showed no homodimerization but formed heterodimers in vitro. Pairwise coexpression of hemagglutinin-and c-Myc epitope-labeled proteins in Arabidopsis cells confirmed the existence of coimmunoprecipitating SPDS1-SPDS2 and SDPS2-SPMS heterodimers in vivo. The epitope-labeled SPDS and SPMS proteins copurified with protein complexes ranging in size from 650 to 750 kD. Our data demonstrate the existence of a metabolon involving at least the last two steps of polyamine biosynthesis in Arabidopsis.

show abstract

“…The CaMV35S promoter of pBI121 vector (Clontech) was replaced by the KDC1 promoter using the HindIII and BamHI sites. This vector was introduced into Agrobacterium tumefaciens LBA4404 by electroporation (Main et al, 1995) and transformed into Daucus carota. To obtain transgenic lines, somatic plantlet hypocotyls were isolated and incubated with the bacteria suspension for 5 min.…”

Section: Plant Transformationmentioning

confidence: 99%

Potassium and carrot embryogenesis: Are K+ channels necessary for development?

et al. 2004

View full text Add to dashboard Cite

The expression pattern of the KDC1 gene, coding for an inwardly-rectifying K(+) channel of Daucus carota , is described in several embryo stages and seedling tissues. Relative quantitative RT-PCR experiments indicated that, during (somatic) embryonic development, the KDC1 transcript appears as early as the globular stage and that the transcript level remains constant throughout the successive heart and torpedo stages. Thereafter, the KDC1 transcript is preferentially expressed in plant roots, but is also present in other tissues, and in particular, in the shoot apical meristem. In situ hybridisation experiments showed that in embryos KDC1 mRNA is detectable preferentially in protoderm cells with a stage dependent expression pattern. At later times, the hybridisation signal is particularly evident in root hairs, root epidermis and endodermis, but is also observed in single cell layers corresponding to L1 of the shoot apical meristem and leaf primordia. Promoter studies with the beta -glucuronidase reporter gene confirm preferential expression of KDC1 in embryo protoderm cells and in plant root epidermis and root hairs. Western blot analysis of embryonic proteins and immunolocalisation experiments on somatic embryos sections revealed the presence of KDC1 during embryo development. Consistent with these observations, patch-clamp experiments performed on protoplasts isolated from embryos at the torpedo stage demonstrated the presence of functional inward rectifying K(+) channels. This is the first report on the expression of a plant ion channel during embryo development.

show abstract

Electroporation Protocols for Agrobacterium

Cited by 21 publications

References 5 publications

Agroinfiltration of grapevine leaves for fast transient assays of gene expression and for long-term production of stable transformed cells

Agroinfiltration of grapevine leaves for fast transient assays of gene expression and for long-term production of stable transformed cells

A Polyamine Metabolon Involving Aminopropyl Transferase Complexes in Arabidopsis

Potassium and carrot embryogenesis: Are K+ channels necessary for development?

Contact Info

Product

Resources

About