Jul 1, 2010

Research Projects

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Developing a plastid transformation technology for maize

(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).

Plastid transformation represents a way of preventing, or severely restricting, the spread of transgenes via pollen. During plastid transformation, new genes are ferried into the DNA of the plastids. In most agricultural crop plants, plastids are inherited maternally, i.e. not via pollen.

Plastid transformation has not yet been successful in cereals, the world’s most important crop plants. This is partly because the selection process that has to be carried out to ensure that all the plant cells are homoplasmatic, i.e. contain only transgenic plastid genomes, is very difficult in cereals. A precursor project has already succeeded in developing a new tissue culture system for maize from leaves with which it is possible to conduct several selection and regeneration cycles. Researchers were able to achieve a high transformation rate of the cell nucleus using this tissue. The aim of this project is to build on these results to develop a transformation technology for maize plastids.

Experiment description

Some of the plastids will be transformed by bombarding cell material (calluses) and leaves of maize plants using a particle gun (or gene gun). The gene constructs used for the transformation contain two marker genes that confer different kinds of antibiotic resistance. This improves the selection of transformed cells. To obtain homoplasmatic plants, the lines are subjected to several selection and regeneration cycles. Key parameters are varied to optimise the procedure:

• Use of a number of different maize lines
• Parameter optimisation in transformation using the gene gun (pressure, distance from sample, etc.)
• Variation of selection conditions using different antibiotic concentrations

The lines in which plastid transformation is successful will be characterised using molecular and genetic tests. At the end of the studies there will be a preliminary biotechnology test application - the integration of genes for Bt toxins into the chloroplast genome of maize.

Results

At the start of the project, various gene constructs were developed and constructed for the transformation of the maize plastids. They contain the possible selection marker genes and a reporter gene (GFP) in various combinations. A gene gun was developed independently and built to enable a more flexible optimization of the various parameters during transformation. Optimization of the parameters is complete.

The experiments for optimizing the selection conditions are also complete, which means that the plastid transformation and selection experiments can begin.