Producing marker-gene-free cereal plants using androgenetic segregation

(2005 - 2008) Institute of Plant Genetics and Crop Plant Research (IPK), Gatersleben

Topic

One way of obtaining marker-gene-free transgenic plants is to transfer two separate DNA sections with effector/marker gene at the same time. Among the sexually produced progeny, those plants can then be identified which, as a result of classic genetic segregation , contain the effector gene but not the selection marker.

Plant breeders require homozygous plant strains to ensure that the strains’ traits are passed on to all progeny, and not just some. However, homozygous plants cannot usually be distinguished from heterozygous plants by their external appearance. This is also true for transgenic plants and means that comprehensive molecular biological tests have to be carried out on the segregating progeny of transgenic plants in order to find homozygous plant lines.

The aim in this project is to simplify the production of marker-gene-free, homozygous plants using androgenetic segregation. The objective is a faster and more efficient production of transgenic barley and wheat plants which are homozygous with regard to their transgene and no longer contain a selection marker.

Information on processes and previous findings:

Experiment description

First, immature embryos of barley and wheat are cotransformed with uncoupled T-DNAs. One T‑DNA contains the effector gene; the other the selection marker. Plants regenerated from these embryos are then tested for transformation efficiency using PCR or DNA hybridisation and those that have integrated both genes in their genome are identified.

In the second step, unripe pollen is isolated from the ears of these plants. To induce androgenetic development, they are subjected to stress treatment. The cells of the pollen’s haploid chromosome set can spontaneously divide, leading to diploidization. The plants regenerated from the pollen cultures (so-called double haploids) are completely homozygous because all the chromosomes have replicated themselves. In the plants that do not spontaneously diploidize (haploid plants) genome replication is induced by treating the plants with colchicine. Colchicine is a cytotoxin obtained from the native autumn crocus.

In the final step, the selection-marker-free transgenic plants are identified using PCR or DNA hybridisation and the system as a whole is assessed with regard to its efficiency and reliability.

Results

Producing the transformation vectors

The project began in mid-2005. In the first step, the binary vectors required for cotransformation were produced. These vectors were then integrated into two different agrobacterium lines. In order to ensure that the effector gene or selection marker is separate in the plant genome after transformation, the two T-DNAs with their genes are arranged differently in the agrobacterium binary vector combinations:

one agrobacterium line containing two vectors, each with one T-DNA
one agrobacterium line containing one vector with both T-DNAs at separate sites
two different agrobacterium lines each containing one vector that contains either the T-DNA with the effector gene or the T-DNA with the selection marker

Gene transfer in immature barley embryos and the production of transgenic plants

The barley was transformed by inoculating immature embryos with the agrobacterium binary vector combinations. The embryos were treated with 14 different agrobacterium lines or mixtures. For each of these 14 treatment variants, three independent experiments were carried out, each with ninety embryos. In total, over 600 plants have been regenerated so far in these experiments which have proved to be transgenic in terms of the selection marker gene. The highest transformation rate was achieved using an Agrobacteria strain containing a binary vector with two separate T-DNAs. With this variant, 270 inoculated barley embryos resulted in 59 lines in which the selection marker gene had been integrated, which corresponds to an efficiency of 21.8%.

Identifying the cotransformed plants
Successful cotransformations were identified by means of PCR using specific primers for the selection marker/effector gene. As expected, the agrobacterium lines and combinations produced different transformation rates. In total, 132 barley plants have been identified so far which have integrated both the selection marker gene and the effector gene in their genome. Most of the co-transgenic plants were also obtained using Agrobacteria in which the two T-DNAs in question were arranged at separate sites on the same vector. In this case, 23 of the 59 transgenic lines generated proved to be co-transgenic, which corresponds to a yield of 8.5 co-transgenic lines per 100 inoculated embryos.

Segregating the T-DNAs and identifying selection-marker-free plants
The method that had been optimised in the first year of the project for obtaining double-haploid (DH) plants from androgenetic pollen cultures was applied to all the cotransgenic barley plants. In this way it was possible to generate DH population for around 80% of the 132 primary co-transgenic lines. The remaining lines are being propagated via self-pollination. These progeny are also being examined for independent segregation of the marker and effector gene. Using these results it will be possible to compare conventional and androgenetic segregation with regards to the production of selection-marker-free plants. PCR was used to detect the presence of the selection marker and the effector gene in the DH lines. In addition, a functional test of the hygromycin resistance conferred by the selection marker was performed using a leaf test, and molecular characterisation of the DH lines was performed using DNA hybridization.

It was found that the two cotransferred T-DNAs had in most cases not been independently segregated in the DH populations and had therefore been integrated together on the same chromosome. In particular, the use of the Agrobacteria line containing a binary vector with two separate T-DNAs has so far failed to produce an independent segregation of marker and effector gene in the DH populations. By contrast, using an Agrobacteria line containing two vectors, each with one T-DNA, produced the highest rate of independently integrated T-DNAs so far. Seven of the eight DH populations obtained were found to have independent segregation of selection marker gene and effector gene, i.e. homozygously transgenic, selection-marker-free plants were identified in each of these seven populations. So far, however, this treatment variant has been carried out with only 90 inoculated embryos, so these results still have to be confirmed with a more meaningful trial size.

The planned trial numbers are currently being made up for all barley treatments. An additional experiment has also begun to test whether the method that has so far proved most effective for barley can also be used with wheat.