Transposition Mediated Re–positioning and Subsequent Elimination of Marker Genes from Transgenic Tomato

Goldsbrough, Andrew P.; Lastrella, Cleofe N.; Yoder, John I.

doi:10.1038/nbt1193-1286

Cited by 80 publications

(70 citation statements)

References 35 publications

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“…Transposon-mediated transgene reintegration can be identified through Southern-blot analysis of transgenic plants using probes specific to the sequences of transposable elements or their donor sites in T-DNA (Goldsbrough et al, 1993;Cotsaftis et al, 2002). However, the Southern hybridization approach is labor intensive for screening a large number of transgenic progeny.…”

Section: Discussionmentioning

confidence: 99%

“…Transposition is an advantageous strategy for marker gene removal, because it allows intact transgene insertion with defined boundaries and requires only a few primary transformants (Cotsaftis et al, 2002). Transposon-mediated transgene reintegration was initially used by Goldsbrough et al (1993) to reposition a Dissociation (Ds) transposon-based GUS reporter gene in transgenic tomato (Solanum lycopersicum). In that study, the Ds(GUS) element in transfer DNA (T-DNA) was tandem linked to both Activator (Ac) transposase (AcTPase) and neomycin phosphotransferaseII (NptII) selectable marker to allow the transposition and segregation of Ds from T-DNA-based AcTPase and NptII.…”

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confidence: 99%

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Efficient Generation of Marker-Free Transgenic Rice Plants Using an Improved Transposon-Mediated Transgene Reintegration Strategy

Gào

Zhou

et al. 2014

Plant Physiol.

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(S.Q.).Marker-free transgenic plants can be developed through transposon-mediated transgene reintegration, which allows intact transgene insertion with defined boundaries and requires only a few primary transformants. In this study, we improved the selection strategy and validated that the maize (Zea mays) Activator/Dissociation (Ds) transposable element can be routinely used to generate marker-free transgenic plants. A Ds-based gene of interest was linked to green fluorescent protein in transfer DNA (T-DNA), and a green fluorescent protein-aided counterselection against T-DNA was used together with polymerase chain reaction (PCR)-based positive selection for the gene of interest to screen marker-free progeny. To test the efficacy of this strategy, we cloned the Bacillus thuringiensis (Bt) d-endotoxin gene into the Ds elements and transformed transposon vectors into rice (Oryza sativa) cultivars via Agrobacterium tumefaciens. PCR assays of the transposon empty donor site exhibited transposition in somatic cells in 60.5% to 100% of the rice transformants. Marker-free (T-DNA-free) transgenic rice plants derived from unlinked germinal transposition were obtained from the T1 generation of 26.1% of the primary transformants. Individual marker-free transgenic rice lines were subjected to thermal asymmetric interlaced-PCR to determine Ds(Bt) reintegration positions, reverse transcription-PCR and enzyme-linked immunosorbent assay to detect Bt expression levels, and bioassays to confirm resistance against the striped stem borer Chilo suppressalis. Overall, we efficiently generated marker-free transgenic plants with optimized transgene insertion and expression. The transposon-mediated marker-free platform established in this study can be used in rice and possibly in other important crops.

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Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

Efficient Generation of Marker-Free Transgenic Rice Plants Using an Improved Transposon-Mediated Transgene Reintegration Strategy

Gào

Zhou

et al. 2014

Plant Physiol.

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“…There are several reasons to produce marker-free plants (Hohn et al, 2001;Hare and Chua, 2002;Miki and McHugh, 2004;Goldstein et al, 2005): marker gene removal can prevent the movement of selectable markers within the environment, simplify the regulatory process and allow the reuse of the same marker. Different methods have been identified that enable marker gene removal: co-transformation (Komari et al, 1996), transposon-dependent repositioning (Goldsbrough et al, 1993), as well as homologous (Zubko et al, 2000) and site-specific recombination (Dale and Ow, 1991). Site-specific marker gene removal will be the main topic of this section.…”

Section: Cre-mediated Excision Of Marker Genesmentioning

confidence: 99%

Elimination of Transgenic Sequences in Plants by Cre Gene Expression

Kopertekh¹,

Schiemann²

2012

Transgenic Plants - Advances and Limitations

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“…However, this approach is not suitable for vegetatively propagated plants such as apple and pear. For these species, the use of transposons [such as the Ac/Ds transposable element system (Goldsbrough et al 1993;Cotsaftis et al 2002) or ipt-type multi-autotransformation vector system (Ebinuma et al 1997a, b;Ballester et al 2007)] or homologous recombination [such as the cre-lox system (Gleave et al 1999;Zuo et al 2001;Cuellar et al 2006;Luo et al 2007) and FLP-FRT system (Kilby et al 1995;Luo et al 2007)] to eliminate the marker gene may work at very low efficiency in apple. That was the case when Schaart et al (2004) were emphasizing systems in which the marker genes are eliminated efficiently soon after transformation by using the cre-lox system.…”

Section: Introductionmentioning

confidence: 99%

Genetic transformation of apple (Malus x domestica) without use of a selectable marker gene

Malnoy

Boresjza-Wysocka

Norelli

et al. 2010

Tree Genetics & Genomes

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Selectable marker genes are widely used for the efficient transformation of crop plants. In most cases, antibiotic or herbicide resistance marker genes are preferred because they tend to be most efficient. Due mainly to consumer and grower concerns, considerable effort is being put into developing strategies (site-specific recombination, homologous recombination, transposition, and cotransformation) to eliminate the marker gene from the nuclear or chloroplast genome after selection. For the commercialization of genetically transformed plants, use of a completely marker-free technology would be desirable, since there would be no involvement of antibiotic resistance genes or other marker genes with negative connotations for the public. With this goal in mind, a technique for apple transformation was developed without use of any selectable marker. Transformation of the apple genotype "M.26" with the constructs pPin2Att35SGUSintron and pPin2MpNPR1 was achieved. In different experiments, 22.0-25.4% of regenerants showed integration of the gene of interest. Southern analysis in some transformed lines confirmed the integration of one copy of the gene. Some of these transformed lines have been propagated and used to determine the uniformity of transformed tissues in the plantlets. The majority of the lines are uniformly transformed plants, although some lines are chimeric, as also occurs with the conventional transformation procedure using a selectable marker gene. A second genotype of apple, "Galaxy," was also transformed with the same constructs, with a transformation efficiency of 13%.

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Transposition Mediated Re–positioning and Subsequent Elimination of Marker Genes from Transgenic Tomato

Cited by 80 publications

References 35 publications

Efficient Generation of Marker-Free Transgenic Rice Plants Using an Improved Transposon-Mediated Transgene Reintegration Strategy

Efficient Generation of Marker-Free Transgenic Rice Plants Using an Improved Transposon-Mediated Transgene Reintegration Strategy

Elimination of Transgenic Sequences in Plants by Cre Gene Expression

Genetic transformation of apple (Malus x domestica) without use of a selectable marker gene

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