Oct 14, 2005

Research Projects

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Elimination of superfluous gene sequences in sugar beet

(2001 – 2004) KWS / PLANTA Angewandte Pflanzengenetik und Biotechnologie GmbH; Einbeck

Topic

This project investigated whether two new systems for the transformation of sugar beet can be used simultaneously to effectively remove superfluous gene sequences, particularly marker genes that are no longer required, from the transformed plant, ideally in one step.

Ac/Ds transposon system. Transposon systems have not yet been used with sugar beet. Tests were therefore carried out to check that the system works in sugar beet.

Further information on methods:

Homologous recombination with the Cre/lox system under the control of a seed-specific promoter. The advantage of this system is the automatic elimination during seed production. The following generation should therefore be marker-gene-free.

Further information on methods:

Summary

Both systems work in principle. However, there are still a few problems to be solved before they can be used in practice. The Ac/Ds transposon system works in sugar beet. It is still unclear how frequent these “gene jumps” are and how far apart the old and new integration sites are in the genome.

Homologous recombination with the Cre/lox system was achieved in tobacco and sugar beet plants with one of the promoters. Another promoter proved unsuitable.

Experiment descripiton

The project consists of several stages and investigations.

Ac/Ds transposon system

  • Producing vector constructs into which the Ac/Ds transposition system is integrated.
  • Testing for functionality using reporter genes which indicate successful repositioning of the genes (= transposition).
  • Improving the transformation efficiency of sugar beet; involving testing various promoters.
  • Stable transformation in sugar beet
  • Analysis of the effect of the transposon using molecular analysis

Homologous recombination with the Cre/lox system

  • Producing vector constructs using the Cre/lox recombinase system
  • Transforming thale cress (Arabidopsis), tobacco or oilseed rape to test various promoters; followed by transformation of sugar beet
  • Testing various promoters that react only in the seed or embryo (seed/embryo-specificity)
  • Molecular characterisation of the transformants using PCR

Results

Both systems work in principle. However, a few problems still need to be sorted out before they can be used in practice.

The Ac/Ds transposon system works in sugar beet. For the first time, it was possible to demonstrate both the excision of the marker gene and its reintegration at a different site in the sugar beet genome.

Progeny of the transgenic sugar beet were also produced.

It is still unclear how frequent the “gene jumps” triggered by the Ac/Ds system are and how far apart the old and new integration sites are in the genome.

Two tobacco shoots. The upper shoot has been transformed using the marker gene elimination system (seed-specific promoter). The desired gene in this case is a gene for a green fluorescence protein (GFP), which is activated only if the marker gene is removed. The lower shoot acts as a non-transgenic control.

Only the upper shoot glows under UV light because the GFP (Green Fluorescence Protein) is activated in this one by the elimination of the marker gene.

Homologous recombination using the Cre/lox system. This system was used to produce forty independent tobacco transformants. Analysis of the following generation (F1) revealed that the seed-specific promoters used vary in their effectiveness.

  • While one promoter resulted in only a few marker-gene-free plants, all the progeny of the tobacco plants with a different promoter were marker-gene-free. In these plants the project aim was achieved.
  • It was possible to demonstrate clearly that these plants really were transgenic by means of the fluorescence effect of the gfp reporter gene. This fluorescence occurs only if the plants are marker-gene-free.

Among tobacco plants, completely marker-gene-free plants were found after two generations.

This result has since been confirmed for sugar beet. Molecular analyses confirm the absence of the marker gene.