Sep 12, 2005

Research Projects

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The development of marker-free gene constructs and their use in microinjection

(2001 – 2004) Fraunhofer Institute for Molecular Biology and Applied Ecology (IME), Department of Genome and Proteome Research, Schmallenberg, Aachen

Topic

Up to now it has not been possible to use microinjection as a method of transformation in plants, because the high internal pressure of the plant cell was destroyed during the injection process, resulting in cell death.

The aim of this project was to examine and further develop methods already tested in preliminary experiments. The idea was that by using microinjection as a new method of transformation, it would be possible to transfer only the functionally required genes in each case, without a marker gene.

Summary

Microinjection was established as a method of transformation in plants. Agroinjection – the injection of specific Agrobacterium proteins together with the constructs – proved a successful strategy here.

Meanwhile, transgenic plants have also been successfully produced without agroinjection. This opens the way to using microinjection commercially as well without great expense.

However, up to now only transgenic plants with a marker gene (kanamycin resistance) have been successfully regenerated, so a marker is currently still essential.

Experiment description

The project involved several stages: First of all, different constructs were prepared for microinjection. Then the plant cells (microcalli) injected by the project partner (the University of Gießen) and the regenerated transgenic plants were characterised using molecular biological techniques.

Preparation of constructs for microinjection

The aim was to prepare constructs that consist solely of the desired gene and the control sequences (promoter and terminator; “clean-gene” fragment). They should contain no additional vector sequences.

To augment the reliability of the procedure and the transformation rate, different variations of the constructs were tested: Based on the “clean-gene” fragments, flanking recognition signals for restriction enzymes were inserted using PCR. The aim was to see whether this increased transformation efficiency.

The left and right “border sequences” from Agrobacterium were similarly inserted into the constructs using PCR.

Development and testing of an agroinjection system

Specific proteins from Agrobacterium tumefaciens, which are involved in the integration of T-DNA into the plant genome, were transferred in parallel with the genes. The aim was to increase the transformation rate.

Two strategies were used to ensure the simultaneous appearance of the “clean gene” fragments and the proteins:

• Using the genes of these proteins in parallel with the constructs. The genes involved in the integration of T-DNA were isolated from Agrobacterium and cloned.
• Injecting the proteins together with the constructs. The proteins were produced recombinantly (in E.coli) and injected together with the T-DNA constructs.

Molecular biological characterisation

Microcalli and plants were regenerated from single cells under selective conditions following agroinjection and characterised using molecular biological techniques. Constructs were initially injected with a marker gene (kanamycin resistance).

To produce transgenic plants without a marker gene, the desired traits were applied by agroinjection without adding the kanamycin gene. To identify the microinjected cells, the non-toxic “Texas Red” dye was inserted in parallel. This remains in the vacuole inside the plant cell for a long time, thereby marking the transformed cell. Cells which did not contain the dye were killed off.

Results

Preparation of constructs for microinjection

A gene for a fluorescent protein (gfp) was used as a reporter, together with a gene which confers resistance to harmful fungi (Verticillum dahliae). The constructs were produced both with and without the marker gene (kanamycin resistance). All genes were placed under the control of the 35S promoter of the cauliflower mosaic virus. The 35S terminator served as the termination signal.

The integrity of all constructs was tested using sequence analysis.

Different variations of the constructs were tested.

Development and testing of an agroinjection system

Of the various strategies used (see above) only agroinjection proved successful. For both genes (gfp and ver) it was possible to establish several independent plants, where molecular analysis of the genome clearly showed the presence of multiple integrations. For each construct several plants were identified which contained the stable, full-length genes and showed expression of these genes.

Molecular biological characterisation

Following successful microinjection of the constructs by the project partner, the University of Giessen, transgenic plants were regenerated from the microcalli and analysed using molecular techniques.

The project was unable to regenerate transgenic plants without markers. Approximately twenty plants per construct were characterised using molecular biological techniques. It became apparent that none of them exhibited the desired transgenes and were therefore wild-type plants. When selective media are not used, transgenic cells can very easily be overgrown by wild-type cells, since these have not been previously damaged by microinjection and can therefore form calli more quickly.

The analysis of additional microcalli and plants also failed to produce positive results, so at present it is still absolutely essential to use a marker.

Meanwhile, in cooperation with the University of Giessen, it has been shown that, with only very slight deviations in the parameters, it is possible to produce transgenic plants without agroinjection. Once these modifications have been verified, it would be conceivable to use microinjection commercially without great expense.