Plastid transformation to prevent the spread of genetically modified plants

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

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.

More from GMO Safety

• SiFo project: Developing a plastid transformation technology for maize