Uptake, integration, expression and genetic transmission of a selectable chimaeric gene by plant protoplasts

Hain, Rüdiger; Stabel, Priska; Czernilofsky, Armin P.; Steinbiß, Hans‐Henning; Herrera-Estrella, Luis; Schell, Jeff

doi:10.1007/bf00330254

Cited by 253 publications

(87 citation statements)

References 36 publications

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“…The BglII-BamHI fragment of pKC7 corresponding to the coding se-| u quence of the npt-II gene was then cloned into the BglII site of pGVL104. The BclI-XhoI fragment in pGVL106 was replaced by the BclI-SalI fragment of pLGVneo2lO3 (12), removing in this way the npt-II leader sequence (24). By reintroducing the BclI fragment of pGVL106 in pGVL118, the plasmid pGVL120 was obtained.…”

Section: Results Nhimentioning

confidence: 99%

Isolation of tobacco DNA segments with plant promoter activity.

Herman¹,

Montagu²,

Depicker³

1986

Mol. Cell. Biol.

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We constructed a promoter probe vector, pGVL120, to isolate plant DNA segments with promoter activity in tobacco. Plant nuclear DNA Sau3A fragments were inserted in front of the npt-II sequence, and a mixture of recombinant plasmids was mobilized to Agrobacterium sp. and used to transform tobacco protoplasts. By kanamycin selection, transformed plant cell lines containing NPT-II T-DNAs were isolated. Eight of these cell lines were regenerated and analyzed for the levels of NPT-H activity in stem, root, midrib, and leaf. These levels demonstrated novel regulation patterns in each isolate. One cell line, T20, was analyzed in detail and found to contain four different T-DNAs. One of the recloned T-DNAs, T20-2, contains an insert of 401 base pairs in front of the NPT-H sequence, and by reintroducing this T-DNA into plant cells we could demonstrate that this insert provides a promoter sequence. The NPT-II enzyme activity under the control of the P20 promoter is especially high in stem and root, but low in leaf and callus, both in the originally isolated T20 plant and in independently isolated transformants with the T20-2 T-DNA.

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Section: Results Nhimentioning

confidence: 99%

Isolation of tobacco DNA segments with plant promoter activity.

Herman¹,

Montagu²,

Depicker³

1986

Mol. Cell. Biol.

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“…Transient expression in protoplasts requires the use of chemical agents (Paszkowski et al, 1984;Hain et al, 1985;Ballas et al, 1987), or of electric pulses (Fromm et al, 1985;Hauptmann et al, 1987) or of both (Shillito et al, 1985) to overcome the uptake control exercised by the plasma-lemma. Although we recommend the use of DMSO, which is assumed to interfere with the membrane (Kawai and Nishizawa, 1984) for optimal uptake, no chemical agents were employed in our initial experiments.…”

Section: Discussionmentioning

confidence: 99%

“…Other plasmids used were pCAP212 (Velten and Schell, 1985), pLGV1103neo (Hain et al, 1985), and pRTlOlcat (Pri~ls et al, 1988b) as well as the WDV-based constructs pWDVneol, pWDVneo2, and pWDVS2 previously described (V. Matzeit, M. Kammann, S. Schaefer, H.-J. Schalk, J. Schell, and B. Gronenborn, manuscript submitted for publication).…”

Section: Plasmids Usedmentioning

confidence: 99%

Uptake and Transient Expression of Chimeric Genes in Seed-Derived Embryos

Gronenborn¹,

Schell²,

Steinbiß³

et al. 1989

The Plant Cell

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Uptake of DNA in dry and viable embryos of wheat by imbibition in DNA solution was detected by monitoring the transient expression of chimeric genes. Gene expression vectors used in this study contained a neomycin phosphotransferase (NPT) II reporter gene fused to various promoters. Some of the chimeric "neo" genes were shown to yield reproducibly NPT II activity in germinating embryos. This NPT II activity was increased markedly When the neo genes were carried by a vector capable of autonomous replication. Dimers of wheat dwarf virus, a monopartite gemini virus, were thus shown to be effective in amplifying the transient expressed NPT II activity in embryos of several cereals. These and other observations indicate that the observed transient expression really results from DNA uptake and expression in plant embryo cells and is not due to contaminating microorganisms.

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“…3 = N. of calli with shoots after 90 days of culture divided by the N. of plated calli. transformation was lower than that expected from transient gene expression in electroporated protoplasts (Hain et al, 1985;Shillito et al, 1985), probably due to some retention of transgene expression ability in damaged protoplasts for periods shorter than 72 h. The chloride ions in buffer I resulted in the production of toxic chlorine gas when electric pulses were applied, leading to a reduction in cellular viability ( Figure 6B). In contrast to the data reported by Senaratna et al (1991), we found that non-electroporated protoplasts incubated with plasmid DNA under the same conditions for longer periods of time never showed transient expression of the reporter gene ( Figure 6C).…”

Section: Effect Of the Electroporation Buffer On Dna Introductionmentioning

confidence: 96%

Factors influencing electroporation-mediated gene transfer to Stylosanthes guianensis (Aubl.) Sw. protoplasts

Quecini¹,

Oliveira²,

Alves³

et al. 2002

Genet. Mol. Biol.

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In order to develop a high-efficiency and reproducible transformation protocol for Stylosanthes guianensis we assessed the biological and physical parameters affecting plant electroporation protoplasts. Energy input, as combinations of electric field strengths discharged by different capacitors, electroporation buffer and DNA form were evaluated. Transformation efficiency was assayed in vivo as transient reporter gene expression, using the GFP-coding gene mgfp5 driven by a CaMV 35S constitutive promoter. Energy input and electric field strength had a critical influence on transgene expression with higher transformation levels being achieved with 250 V.cm -1 discharged by 900 and 1000 µF capacitors. Linear plasmid DNA, the absence of chloride and the presence of calcium ions also increased transient gene expression, albeit not significantly.

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Uptake, integration, expression and genetic transmission of a selectable chimaeric gene by plant protoplasts

Cited by 253 publications

References 36 publications

Isolation of tobacco DNA segments with plant promoter activity.

Isolation of tobacco DNA segments with plant promoter activity.

Uptake and Transient Expression of Chimeric Genes in Seed-Derived Embryos

Factors influencing electroporation-mediated gene transfer to Stylosanthes guianensis (Aubl.) Sw. protoplasts

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