Dec 10, 2009

Research Projects

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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 biomolecular tests have to be carried out over several generationsto find homozygous plant lines.

The production of marker-free, homozygous transgenic barley and wheat plants was simplified in this project by using so-called double haploid plants. These were generated from unripe pollen, which can produce plants under suitable culture conditions. These plants are homozygous.

Summary

A practical and efficient method was developed for generating marker gene-free, homozygous plants from unripe transgenic barley pollen. The desired independent segregation of selection markers and target gene was seen in seven double haploid barley populations.

This approach for producing marker-free, homozygous transgenic plants could also be applied, for example, to wheat.

Experiment description

First, immature embryos of barley and wheat were cotransformed with uncoupled T-DNAs. One T-DNA contains the effector gene; the other the selection marker. Plants regenerated from these embryos were 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 was 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) were completely homozygous because all the chromosomes have replicated themselves. In the plants that do not spontaneously diploidize (haploid plants) genome replication was 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 were identified using PCR or DNA hybridisation and the system as a whole was assessed with regard to its efficiency and reliability.

Results

Producing the transformation vectors

Initially, the gene to be integrated was transferred to the bacterial vectors required for the co-transformation. Subsequently, these vectors were integrated into two different agrobacterial lines. The aim was to develop a method with which the target and selection marker genes were separated (on different chromosomes) in the plant genome in as many cases as possible. Different combinations of bacterial strains and vectors were tested.

• Regime 1: Two agrobacterial lines were mixed, each containing one vector that comprised either the T-DNA with the target gene or that with the selection marker.
• Regime 2: One agrobacterial line contained two vectors, one for each T-DNA.
• Regime 3: One agrobacterial line contained one vector in which both T-DNAs were located at separate sites.

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

The transformation of barley was carried out by inoculating unripe pollen with agrobacteria. These transferred both of the uncoupled T-DNAs with the target gene or the selection marker to the regenerative cells. The embryos were treated with 14 different agrobacterial lines or their combinations. For each of these 14 treatment variations, three independent trials, each with 90 embryos were performed. In total, 606 plants carrying the selection marker were generated in these experiments.

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 57 lines in which the selection marker gene had been integrated, which corresponds to an efficiency of 21.1%.

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, 128 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, 24 of the 57 transgenic lines generated proved to be co-transgenic, which corresponds to a yield of 8.9 co-transgenic lines per 100 inoculated embryos.

Segregating the T-DNAs and identifying selection-marker-free plants

This method, optimised at the start of the project, for preparing double haploid (DH) plants from embryo gene pollen culture was used on all co-transgenic barley plants.

In this way, viable DH plants could be generated for 107 of the 128 primarily co-transgenic lines (83.6%). In the DH lines the presence of selection markers and target genes was determined using PCR. In addition, the functional resistance against the antibiotic hygromicin mediated through the selection marker was investigated using a leaf assay, and a molecular characterisation of the DH strain was performed using DHA hybridisation.

It was shown that mostly both co-transferred T-DNAs in the respective DH populations were not independently segregated and were, therefore, integrated on the same chromosome. However, using one agrobacterial strain containing two vectors, each with just one T-DNA, the highest rate of uncoupled integrated T-DNA found until now was demonstrated. Seven of the nine DH populations containing these variants showed independent segregation of the selection maker and the target gene, i.e. homozygous transgene, selection marker-free plants were identified in each of these seven populations. Based on 100 barley embryos inoculated with agrobacteria, this demonstrates an efficiency of 2.6% for the preparation of homozygous transgenic, selection marker-free strains. Therefore, the method developed in the framework of this project proved to be very promising for use in practice. Finally, in an additional experiment, it was also shown that, in principle, this method developed with barley can also be used with wheat to produce selection marker-free, homozygous transgenic plants.