Jul 1, 2010

Research Projects

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Plastid transformation to prevent the spread of genetically modified plants

(2008 – 2011) Max Planck Institute of Molecular Plant Physiology Potsdam

Topic

When it comes to assessing the safety of transgenic plants, the possibility of the new genes being transferred by pollen or seeds plays an important role. Strategies are therefore being developed around the world to prevent gene transfer via pollen or seeds (biological confinement).

This project aims to refine an existing confinement system for tobacco. This is a system that uses plastid transformation. During plastid transformation, new genes are ferried into the DNA of the plastids. Conventional methods insert new genes into the DNA of the cell nucleus. Most agricultural crop plants inherit plastids maternally, i.e. not via pollen. Plastid transformation therefore presents a possible way of preventing, or severely restricting, the spread of genetically modified plants.

Preliminary studies for this project found two processes that might impair the safety of this method:

• occasional paternal inheritance (paternal leakage) of plastids
• gene transfer from the plastid genome to the nuclear genome of the plants.

Both events are extremely rare in the greenhouse under standard conditions. However, in normal growing conditions, plants are regularly exposed to stress situations. One of the project aims is therefore to investigate the impact of environmental conditions on these safety-related processes. The idea is also to develop a strategy to prevent the expression of transgenes in the cell nucleus.

Experiment description

Paternal plastid inheritance – dependence on environmental factors

Tobacco lines will be produced that contain a spectinomycin-resistance gene (aadA) and a reporter gene (gfp) in the plastid genome. These plants will be exposed to various stress factors (heat stress, drought, light stress and cold stress) during pollen formation. The pollen will then be used to pollinate male-sterile conventional lines. If the plastids are inherited via the pollen, the inherited resistance will cause the progeny to display green leaf areas on a medium containing spectomycin, as well as non-resistant white areas. These leaf areas glow under UV light because the plastids contain the gfp gene as well. The rate of inheritance will be determined by counting the seedlings. The progeny will then be examined using molecular biological methods (PCR).

Gene transfer to the nucleus – dependence on environmental factors

Tobacco lines will be produced that contain two antibiotic-resistance genes in the plastid genome: the aadA gene with plastid-specific signals, and a kanamycin-resistance gene (nptII) that has nucleus-specific signals. This means that the nptII gene can only be expressed in the nuclear genome, and not in the plastid genome.

Leaf samples from these plants will be exposed to various stress factors (heat stress, drought, light stress and cold stress). These leaf samples will be able to produce new plantlets on a regeneration medium in the presence of kanamycin only if the plastid genome has been transferred to the nuclear genome. The number of regenerated plants will provide information about the transfer rate.

Strategy for preventing the expression of a transgene in the nuclear genome

When genetic information is transcribed into proteins, certain sequences (introns) must be removed from the RNA (splicing). The splicing process requires splicing factors. Introns from the plastid and nuclear genome do not have the same construction and need different splicing factors. In the method described here, plastid intron sequences are integrated into a plastid transgene. If plastid DNA does find its way into the cell nucleus, the absence of the right splicing factors will prevent the expression of the transgene.

To test this strategy, plastid intron sequences will be added to the nucleus-specific kanamycin-resistance gene and transformed in plastids. Leaf samples from the transformed plants will then be exposed to various stress factors and selected using kanamycin as described above.

Results

Dependence of paternal plastid inheritance on environmental factors

To determine the dependence of paternal plastid inheritance on environmental conditions, the stress conditions first had to be optimized so that they did not interfere too much with pollen formation, but still achieved a sufficiently strong stress effect. This part of the research is complete.

Transgenic tobacco lines have since been produced and exposed to the optimized stress conditions. Preliminary cross-fertilization experiments were conducted between the tobacco lines and a male-sterile conventional line. The seeds of these plants were harvested and then selected on a medium containing spectomycin. These results are currently being evaluated and confirmed statistically through further comprehensive selection experiments.

Dependence of gene transfer to the nucleus on environmental factors

Before determining the dependence on environmental factors of gene transfer to the nucleus, the various stress conditions were optimized. The optimization stage is now complete.

Transgenic tobacco lines have been produced containing two antibiotic-resistance genes in the plastid genome. Leaf samples from these plants were exposed to the optimized stress conditions. The first gene transfer lines were successfully regenerated under stress conditions on a medium containing kanamycin. The effect of the various stress conditions on the gene transfer rates from the plastid genome to the nuclear genome is currently being investigated.

Strategy for preventing the expression of a transgene in the nuclear genome

First, a gene construct was produced with plastid intron sequences. The intron sequences were then combined with a kanamycin-resistance gene to form another gene construct. The first plastid transformation experiments with this construct have already been successfully conducted.