Nov 15, 2010

Research Projects

Send

How reliable is the confinement of transgenes in the plastids of genetically modified plants?

(2008 – 2011) Department of Biology I, Biology Faculty, Ludwig-Maximilians-Universität München

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 plastid transformation as a confinement system. The focus is on researching the molecular genetic background to the (undesirable) outcrossing of plastid genes.

Further information on methods:

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.

Experiment description

Transplastomic petunia lines that contain a marker gene in their plastid genome…

…are dusted with pollen from petunia lines that present mutations in their nuclear genome. The mutations are caused by ‘jumping genes’. The activity of these jumping genes can be identified through the red areas that form in the flower.

A large number (450) of petunia lines (F2) is being produced that contain both the marker gene (inherited from the maternal side) and the nuclear mutations. When individual mutants have a high paternal plastid inheritance, this can be detected in the cross-bred progeny using molecular biological methods. These plants will contain the marker gene.

Outcrossing experiments in the field

A: Pollen donor plant (father). The plastids (red) are equipped with a marker gene.

B: Recipient plant (mother), wild type line with unmodified plastids (green)

C: Progeny of wild type line. A plastid (red) has been inherited via the pollen.

Identifying mutants with increased paternal plastid inheritance

In plants, inheritance of plastid DNA is normally exclusively maternal. This is because during pollen development, the plastid DNA in the generative pollen cells is broken down. This process probably does not occur spontaneously; it is probably subject to genetic factors. Using mutants that display a higher frequency of paternal plastid inheritance, the project will attempt to identify and investigate these genetic factors. Once these factors are known, it will be possible in the long term to produce plants in which these genetic factors have been modified in such a way that the ‘pollen transfer of plastid DNA’ event can be reduced still further.

Outcrossing experiments

In order to detect the (undesirable) event of pollen transfer of plastid DNA, cross-breeding experiments are being conducted in collaboration with the team at the University of Rostock.

The cross-breeding experiments are being conducted with the model plants petunia and Arabidopsis, since transplastomic oilseed rape and maize lines will not be available until towards the middle/end of the research project.

The recipient plants are wild type lines (mother plants). Pollen donor lines are planted next to them. These lines are equipped with marker genes. The Arabidopsis lines have a natural herbicide resistance that has been produced via point mutation in the plastid genome. The petunia lines are genetically modified and carry an antibiotic-resistance marker gene in their plastid genome (streptomycin/spectinomycin).

Phenotypic checks are carried out on the cross-bred progeny of the wild type lines to check for the presence of the marker genes. If the marker gene is detected in the progeny, then plastid DNA has been transferred via the pollen.

Outcrossing experiments and quantification of the undesirable spread of plastid DNA in oilseed rape are planned for the third year of the project.

The outcrossing experiments are being conducted under greenhouse and field conditions at the Agrobiotechnikum centre for agrobiotechnology research in Gross Lüsewitz under the leadership of Prof. Broer’s team from the University of Rostock.

Results

Identifying mutants with increased paternal plastid inheritance

Petunias: At the start of the project there were 450 transplastomic lines (F2) in which nuclear mutations had been induced. For each F2 line, six progeny produced by selfing are crossed as pollen donors with wild type plants as the pollen recipients. The resulting cross-bred progeny are being examined for the presence of the transgene.

300 lines containing nuclear mutations have so far been examined and two possible mutants have been identified. The presence of the transgene was detected in their progeny using PCR. These mutants are currently being analysed.

Arabidopsis: At the start of the project, there were 10,000 Arabidopsis lines in which nuclear mutations had been induced that have a point mutation in the plastid genome making them resistant to the herbicide atrazine. Since the start of the project, 1200 of these mutants have been crossed with the wild type plants, using the mutants as the pollen donors. All the cross-bred progeny were tested for resistance to atrazine. So far, no mutants with an increased paternal plastid inheritance have been identified.

Outcrossing experiments in the field

Petunias: The analyses of the progeny from the petunia crosses are not yet complete.

Arabidopsis: In a field trial in Gross-Lüsewitz, Arabidopsis lines with a point mutation in the plastid genome (herbicide-tolerant pollen donors) were crossed with Arabidopsis wild type lines. The progeny were harvested and 1.2 million plants were examined for herbicide resistance. Herbicide resistance was not detected in any of the plants.