DEVELOPMENT OF MITOCHONDRIAL TRANSFORMATION IN PLANTS TOWARD CREATING ELITE HYBRID WHEAT

The major goal of this Phase I project is to provide an efficient method to gene edit plant mitochondrial DNA, i.e., wheat mitochondrial DNA. Achievement of this goal would enable future production of desirable phenotypes in plants by expression of new genes in plant mitochondria. In particular, a subsequent Phase II goal would be use of the Phase I technology to create cytoplasmic male sterile ("CMS") lines of wheat. CMS wheat lines would allow for the creation of hybrid wheat lines, which would result in higher wheat yields. Specific objectives of this Phase I project are the following: (1) Identify sequences useful for maintaining exogenous DNA in plant mitochondria. DNA sequences from diverse organisms will be assayed for their ability to promote DNA replication in plant mitochondria. This capability will be useful to maintain episomal plasmids in plant mitochondria. These episomal plasmids will be intermediates for the subsequent transformation of plant mitochondrial DNA. (2) Identify sequences that convey mitochondrial-specific gene expression. DNA sequences will be assayed for their ability to promote mitochondrial-specific gene expression in plant mitochondria. Candidate DNA sequences include, but are not limited to, sequences from plant mitochondria required for transcription initiation and termination, translational efficiency, codon usage and intron splicing.. Mitochondrial-specific regulatory elements will be useful for the current Phase I goal of gene editing in plant mitochondria. (3) Develop a screenable marker that is only expressed in mitochondria. Gene(s) encoding fluorescent proteins will be modified to allow for specific expression in plant mitochondria. Modifications will include genetic elements identified above for mitochondrial-specific gene expression. An easily assayable screenable or selectable marker gene that is specific for plant mitochondria is currently lacking. Development of such a screenable marker gene will facilitate transformation of plant mitochondria. (4) Use CRISPR technology to gene edit wheat mitochondrial DNA. Our recently published method used CRISPR technology to facilitate gene editing of yeast mitochondria and algal chloroplasts. In Phase I of this proposal we will extend this technology to plant mitochondria. Our primary target will be wheat mitochondria.