Production of marker-gene-free barley plants by means of outcrossing after co-transformation

(2001 - 2004) Max Planck Institute (MPI) for Plant Breeding Research, Cologne

Topic

The purpose of the research was to develop a system whereby target and marker genes are incorporated into the plant genome independently of each other by means of agrobacterium -mediated transformation (co-transformation).

A suitable co-transformation system is a precondition for eliminating the marker gene in the progeny of the transformed plant by means fo segregation .
A further aim is to prevent unwanted DNA from being transferred during agrobacterium-mediated transformation.

Summary

Using comparative investigations, it was possible to demonstrate in this project that plants can be produced without selection markers in a very simple way by means of outcrossing followed by PCR analysis.

Segregation can be achieved in a very simple way by carrying out the co-transformation with two agrobacterium strains, each with one plasmid with the target gene or marker gene. The special feature of this approach is that one can use the same strain of bacteria with an optimum selection marker for every transformation – even with different target genes. This removes the need for costly cloning of the plasmids. The system is utilizable immediately.

Experiment description

Transgenic barley plants in the greenhouse

Representation of the tissue culture stages to obtain a shoot

Work was carried out on the development of suitable binary vectors. At the same time, conditions were established under which these vectors can also be used for transformation of monocotyledons.

Research project: Optimization of vectors for the production of marker-gene-free plants

Two co-transformation approaches were compared: one agrobacterium strain with one plasmid bearing two T‑DNAs , and two agrobacterium strains each with one plasmid (one with the target gene and one with the selection gene).

The transgenic plants were then ripened in the greenhouse for segregation analysis (self-fertilization, cross-pollination). Molecular biological tests were carried out on the next generation to establish how frequently transgenic plants occur without a selection gene. The plants were also subjected to molecular biological analyses (PCR , Southern hybridisation ).

Embryo Rescue was used with the aim of accelerating the breeding of the next generation.

Results

Barley of the Golden Promise variety was transformed using both co-transformation methods. More than one hundred transgenic plants were tested. Both methods resulted in segregation, i.e. the PCR method found plants without a selection gene but with the target gene.