Aug 26, 2010

Research Projects

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Quantifying adventitious outcrossing of plastid genes

(2008 – 2011) Department of Agrobiotechnology - Institute of Land Use (ILN) - Faculty of Agricultural and Environmental Sciences - University of Rostock

Topic

New research projects are focusing on genetic modification of plants in the plastids rather than in the cell nucleus. Plastids are small units of a plant cell that have their own DNA. One of the advantages of plastid transformation is that the genetic information in the plastids is not passed on via pollen, which means that a potential spread of the inserted genes can be prevented and the genome can be contained within the plant (biological confinement).

The aim of this project is to test the reliability of this kind of confinement system. Since there are indications that the plastid confinement system may be ‘leaky’, the project will assess the frequency of (adventitious) gene transfer of plastid DNA via pollen in field trials using the model plants petunia and Arabidopsis.

Experiment description

Plastid transformation will represent a suitable method for containment only if the transgenes transferred to the plastid genome are passed down much less frequently to the next generation via pollen over the long term.

Crossing experiments with petunia, Arabidopsis and oilseed rape

Since transplastomic oilseed rape and maize lines will not be available until part-way through the research project, in the first two years of the project, field trials to investigate the frequency of pollen-mediated transfer of plastid DNA will be conducted on the model plants petunia and Arabidopsis. Greenhouse trials with oilseed rape are planned for the third year.

The trials are designed in such a way that pollen transfer events involving plastid DNA can be detected with a high level of accuracy. To this end, strip-cropping will be used with donor and recipient plants next to each other.

The petunia donor lines have been genetically modified. They have marker genes (antibiotic resistance) and a reporter gene (GUS gene) in their plastid genome. Conventional lines are used as pollen recipients. If the reporter gene is detected in the progeny of the conventional lines, this will show that plastid DNA has been transferred via the pollen.

To detect gene transfer, the seeds of the recipient plants are harvested, grown in the greenhouse and tested for the presence of the reporter gene using a colour test (GUS test). The plan is to test seeds on a large scale (at least 300,000) in order to be able to make statistically secure statements about the probability of (adventitious) pollen transfer of plastid DNA.

The project is part of the CONFICO joint project on “Developing and testing plastid transformation as a confinement system in oilseed rape and maize”. The project partners are the Department of Botany (Prof. Dario Leister), the Cell Biology and Cell Culture group (Prof. Hans-Ulrich Koop) of the University of Munich, the Chair of Genetics (Prof. Alfons Gierl) of Technische Universität München and the Chair of Agrobiotechnology (Prof. Inge Broer) of the University of Rostock.

Results

Arabidopsis thaliana: The first field trial with Arabidopsis took place in late summer 2008 and was repeated using the same experiment design in June 2009. The plots were planted alternately with one or three rows of the herbicide-tolerant Arabidopsis donor lines and one row of each of the male-sterile recipient lines.

The quantity of seeds on the recipient plants was measured after harvest. In 2009 the harvest came to 1,209,190 seeds – only a quarter of the number harvested in 2008 (4,655,477). The lower quantity of seeds was probably due to the much faster ripening of the Arabidopsis plants in the higher temperatures of 2009. The seeds are currently being analysed by a project partner (AG Leister).

Petunias: Because of delays in obtaining the release authorisation, the 8670 petunias were not planted until August in 2009.

The trial design was the same as for Arabidopsis in 2008 and 2009. The lilac-coloured transplastomic lines (T16) were the pollen donors. The white conventional lines (W115) were the pollen recipients. On the control field, the isogenic variety (Pink Wave) was the pollen donor and the same conventional line was the recipient.

In 2009, a total of 12,400 flowers were marked. The petals and stamens were removed from the female flowers, which were then pollinated by hand with the transgenic petunias. 1440 seed capsules were harvested, representing around 900,000 seeds.

The seeds were grown in the greenhouse in the dark at temperatures of 18-24 °C to produce seedlings. A GUS colour test is currently being conducted to assess whether paternal plastid inheritance has taken place. If the seedlings present blue patches, molecular biological methods (PCR) are used to assess whether the plant does in fact contain the transgenic gene sequence.