Sep 26, 2005

Research Projects

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Optimisation of vectors for the production of marker-gene-free plants

(2001 – 2004) Federal Centre for Breeding Research on Cultivated Plants (BAZ); Institute of Grapevine Breeding, Geilweilerhof, Siebeldingen

Topic

Binary vectors are used for the Agrobacterium-mediated transformation of plants. The aim was to optimise them in such a way that only desired DNA sequences are transferred to the plants.

Consequently the objectives were as follows:

• To develop suitable binary vectors which can be used for the co-transformation of two independent genes.
• To integrate only the desired T-DNA (more precise integration – termination accuracy – of T-DNA at the left border) This would overcome the problem of the “leaky” left border. It has long been known that the transfer and integration of T-DNA starts at the RB (right border) and ends at the LB (left border). But it would appear that the LB is “leaky”, since sequences outside the T-DNA are frequently transferred as well.
• To reduce the size of binary vectors. This is necessary to facilitate the transfer of large T-DNA segments, possibly with several genes.

Summary

It appears that co-transformation with one vector and two independent T-DNAs offers the best chance of success. It has not been possible within the lifespan of this project to determine whether subsequent generations would contain plants that carried only the beneficial gene.

Vectors were developed which should facilitate a more accurate integration of the T-DNA.

An appreciably smaller vector has been constructed (less than 5 kb). Work on the relevant Agrobacterium strain is still continuing.

Experiment description

First of all, new binary vectors were developed which seemed capable of fulfilling the objectives.

• These vectors were tested on the model plant Arabidopsis using Agrobacterium-mediated transformation.
• The functionality of the vectors was subsequently examined by conducting molecular biological analyses of the transformed plants (PCR, Southern hybridisation). In addition, various crosses and self-fertilisations were carried out (segregation analysis).

In co-operation with another sub-project of the research group, vectors have been developed which can also be used with monocotyledons such as barley.

Results

The first generation of vectors suitable for co-transformation of two genes is currently undergoing testing.

• It appears that co-transformation with one vector and two independent T-DNAs offers the best chance of success. The vectors contain two independent T-DNAs, each with their own start and stop sequence (right border and left border). In between is a section of DNA which acts as a spacer to ensure that the T-DNAs are integrated in the genome independently of one another and not in the same place.
• 33 transformed Arabidopsis plants underwent PCR analysis to investigate the presence of the two T-DNAs separated by the spacer. The T-DNA with the selection marker was detected in all the plants under investigation. In eleven of those plants the second T-DNA with the useful gene was also detected. No spacer was found, which indicates that the T-DNAs had integrated independently of one another. Within the scope of this project it was not possible to determine conclusively whether, in subsequent propagation, plants could be selected which carry just the useful gene and not the selection gene (segregation analysis).

Vectors were developed which should facilitate a more accurate integration of the T-DNA. The left border, i.e. the termination sequence, was doubled up, sometimes even quadrupled. To date, 30 transformed Arabidopsis plants have been studied. DNA sequences from beyond the left border were transferred into the transgenic plant in only one case. It was not possible to carry out segregation analyses for statistical confirmation before the project ended.

In the part of the experiment concerned with reducing the size of the vector, it was possible to reduce the vector by approximately half (to below 5 kb: 4.8 kb with the selection marker, 3.3 kb without). It was possible to remove sequences which did not have to be located on the plasmid. In this case, the information in the partition genes must be present in the genome of the Agrobacterium instead of on the plasmid. However, the integration of these genes into the genome of the Agrobacterium proved relatively difficult and could not be completed within the lifespan of the project.