Gene delivery in plant mitochondria
We have developed a system for transport of genetic information into the plant mitochondria based on a multi-domain protein expressed from the nucleus, engineered to recruit RNA molecules and transport expression units into the organelle. Reverse transcription can be achieved subsequently by expression of a recombinant enzyme with reverse transcription activity, which is specifically targeted to the mitochondria. Integration of the cDNA at specific sites is achieved by homologous recombination.
The ability to transfer genetic information into the plant mitochondria opens new possibilities for crop improvement. One of the most important plant trait associated with the mitochondrial genome is cytoplasmic male sterility (CMS), manifested as the inability to produce viable pollen. It is a much sought after trait in agriculture because it facilitates hybrid seed production without the need for mechanical or chemical sterilisation. Hybrid plants have become increasingly important in various commercial crops around the world as their yield is substantially higher than conventional cultivars.
Molecular analyses of mitochondrial genomes associated with CMS showed that it is associated with mitochondrial DNA rearrangements. Such abnormal mitochondrial gene organisation has been associated with CMS in various plant species including petunia, maize, rice, sunflower and common bean. Sequence analyses showed that the rearrangements of mitochondrial genes observed in CMS plants create a novel open reading frame (novel gene) encoding a protein that is interfering with pollen development. Production of CMS lines using classical breeding methods is a long and tedious process that takes many years to achieve because it requires repeated back crossing to fix the desirable traits in order to eliminate the unwanted genetic background.
We have shown that it is possible to transfer the CMS-inducing PCF open reading frame encoded by petunia mitochondria, using our mitochondria gene transfer method, to tobacco mitochondria and produce male sterile tobacco lines.
We are currently developing a system that allows the expression of the different components required for mitochondria transformation transiently without the need for prior transgene integration in the nucleus, the aim is to be able to modify the mitochondrial genome at precise locations to integrate CMS-inducing loci in the mitochondria of elite varieties and at the same time avoiding any nuclear transgene integration. We are using the well characterised CMS locus orf79 from rice mitochondria as a model for our experiments in wheat, corn and rice.