A transformation method for obtaining marker-free plants of a cross-pollinating and vegetatively propagated crop

Vetten, Nick de; Wolters, Anne‐Marie A.; Raemakers, Krit; Meer, Ingrid van der; Stege, Renaldo ter; Heeres, Els; Heeres, P.; Visser, Richard G. F.

doi:10.1038/nbt801

Cited by 221 publications

(122 citation statements)

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“…Formation of genetic chimeras have also been reported when LBinitiated T-DNA ligated back to a RB-initiated T-strand (De Buck et al, 1999). The not correct initiating, processing and ending of the T-region during transformation of plant cells using Agrobacterium-mediated transformation method often results in integration of extra backbone DNA originating from the binary plasmid in the plant genome (de Vetten et al, 2003). By using only the RB and reducing the binary PROGMO vector backbone DNA, the complete vector will be integrated in the plant genome as one large T-DNA.…”

Section: Discussionmentioning

confidence: 99%

“…De Vetten et al (2003) reported transformation of potato without the use of selectable markers. The best results were obtained with the potato variety Karnico using the Agrobacterium tumefaciens strain AGL0 that exhibits extremely high transformation efficiency because it contains a DNA region originating from a super virulent A. tumefaciens strain.…”

Section: Introductionmentioning

confidence: 99%

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Generation of Marker- and Backbone-Free Transgenic Potatoes by Site-Specific Recombination and a Bi-Functional Marker Gene in a Non-Regular One-Border Agrobacterium Transformation Vector

2006

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A binary vector, designated PROG-MO, was constructed to assess the potential of the Zygosaccharomyces rouxii R/Rs recombination system for generating marker-and backbone-free transgenic potato (Solanum tuberosum) plants with high transgene expression and low copy number insertion. The PROGMO vector utilises a constitutively expressed plant-adapted R recombinase and a codA-nptII bi-functional, positive/negative selectable marker gene. It carries only the right border (RB) of T-DNA and consequently the whole plasmid will be inserted as one long T-DNA into the plant genome. The recognition sites (Rs) are located at such positions that recombinase enzyme activity will recombine and delete both the bi-functional marker genes as well as the backbone of the binary vector, leaving only the gene of interest flanked by a copy of Rs and RB. Efficiency of PROGMO transformation was tested by introduction of the GUS reporter gene into potato. It was shown that after 21 days of positive selection and using 300 mgl -1 5-fluorocytosine for negative selection, 29% of regenerated shoots carried only the GUS gene flanked by a copy of Rs and RB. The PROGMO vector approach is simple and might be widely applicable for the production of marker-and backbone-free transgenic plants of many crop species.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Generation of Marker- and Backbone-Free Transgenic Potatoes by Site-Specific Recombination and a Bi-Functional Marker Gene in a Non-Regular One-Border Agrobacterium Transformation Vector

2006

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“…Overexpression of the endogenous Atwbc19 ABC transporter gene confers kanamycin resistance (Mentewab and Stewart 2005). For easyto-transform crops such as potato, selectable marker genes are unnecessary (de Vetten et al 2003). Such crops can be cultivated with Agrobacterium carrying an intragenic vector with an inserted gene-of-interest, followed by the regeneration of plants that are screened via PCR to find the transformants.…”

Section: Considerations For the Proper Design Of Intragenic Vectorsmentioning

confidence: 99%

Intragenic vectors for gene transfer without foreign DNA

et al. 2006

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The intragenic vector system involves identifying functional equivalents of vector components from the genome of a specific crop species (or related species to which it can be hybridised) and using these DNA sequences to assemble vectors for transformation of that plant species. This system offers an attractive alternative to current genetic engineering strategies where vectors are based on DNA sequences that usually originate from bacteria. The construction of intragenic vectors enables the well-defined genetic improvement of plants with all transferred DNA originating from within the gene pool already available to plant breeders. In this manner genes can be introgressed into elite cultivars in a single step without linkage drag and without the incorporation of foreign DNA. The resulting plants are non-transgenic, although they are derived using the tools of molecular biology and plant transformation. The use of intragenic vectors for the transfer of genes from within the gene pools of crops may help to alleviate some of the major public concerns over the deployment of GM crops in agriculture, notably the ethical issue associated with the transfer of DNA across wide taxonomic boundaries. This paper reviews the progress toward the development and use of intragenic vectors and the implications of their use for the genetic improvement of crops.

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“…With our MDTT, we were able for the first time in tree fruit crops to regenerate marker-free transgenic plants without the need for sexual crossing, repeated transformation, or selectable markers. There is a report of a low efficiency transforma- nt not tested tion system for the production of marker-free potato plants in which the use of a selectable marker was omitted (de Vetten et al 2003). In our case, the efficiency of transformation was 12-25% depending on the apple cultivar used.…”

Section: Discussionmentioning

confidence: 68%

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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A transformation method for obtaining marker-free plants of a cross-pollinating and vegetatively propagated crop

Cited by 221 publications

References 20 publications

Generation of Marker- and Backbone-Free Transgenic Potatoes by Site-Specific Recombination and a Bi-Functional Marker Gene in a Non-Regular One-Border Agrobacterium Transformation Vector

Generation of Marker- and Backbone-Free Transgenic Potatoes by Site-Specific Recombination and a Bi-Functional Marker Gene in a Non-Regular One-Border Agrobacterium Transformation Vector

Intragenic vectors for gene transfer without foreign DNA

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

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