After selection of transgenic plants the presence of the selectable marker, which is no longer required, can pose both regulatory and technical problems. Therefore an easy and versatile system for removal of all unnecessary DNA sequences from transgenic plants is desirable.

Existing technologies are time consuming, require additional handling and are often inefficient. In our approach a genetic program is introduced together with the Gene Of Interest (GOI) which allows the selection of homozygous transformants and removes the selectable marker effectively, without any extra handling and this in the same time frame as compared to conventional transformation protocols in which the marker is not removed. In this way the removal of the marker can perfectly coincide with the analysis of transgenic plants.

The genetic program consists of three transcriptional units, placed between tandemly oriented recognition sequences for a site-specific recombinase. A first unit comprises the site-specific recombinase gene, which is placed downstream of a germline-specific promoter. The second and third unit contain a positive and negative selectable marker respectively. We developed and tested two modules of this genetic program. In the first module a two-germline-specific promoter is used. In the second module we used a single-germline-specific promoter.

The choice of which module to use depends on a number of parameters such as available space, cost aspects, etc . Using the first module marker-free first sexual progeny could be obtained. Losing the marker at this stage meant it could not be used to screen for single-locus inserts and homozygous plants. This information can potentially be obtained by screening numerous plants by PCR and qPCR (25% of the analyzed plants meet these criteria). Once marker-free transgenic plants are identified up scaling of the seeds can be done. Using the second module, the marker was removed in only one germline. This allowed the use of the marker in the first sexual progeny; a 1:1 segregation indicated single locus integration. Plants surviving the selection and containing a recombined and an unaltered allele are homozygous for the GOI. These plants could easily be discriminated for by standard PCR (50% of the analyzed plants met these criteria). Second sexual progeny seeds of these plants were sown on medium allowing counter selection of the negative selectable marker. Only single locus, homozygous and marker-free plants survived. These constitute 50% of the sown seeds each rendering a marker-free and homozygous seed stock. Moreover the introduction of the gfp gene placed under control of a promoter active in seeds would allow to select for marker-free second sexual progeny seeds and to separate them from non-marker-free seeds without the need for germination on counter selective medium. In this way seed stocks of single locus homozygous marker-free transgenic plants can be obtained in the same generation time as in module 1 but with less effort and the need for the costly qPCR to identify homozygous plants.

This flexibility allows one to choose the most suited module case by case to obtain marker-free transgenic plants effectively, without extra handling and without losing valuable time. Results obtained by using both modules in A. thaliana will be presented.
Original languageEnglish
Title of host publicationAbstract book Knowledge for growth, June 8 2007, Ghent-Belgium
Publication statusPublished - 8 Jun 2007
EventFinds and Results from the Swedish Cyprus Expedition: A Gender Perspective at the Medelhavsmuseet - Stockholm, Sweden
Duration: 21 Sep 200925 Sep 2009

Conference

ConferenceFinds and Results from the Swedish Cyprus Expedition: A Gender Perspective at the Medelhavsmuseet
CountrySweden
CityStockholm
Period21/09/0925/09/09

    Research areas

  • Marker-free transgenic plants, site-specific recombinase

ID: 1612355