This project proposed to increase grain yield in wheat by manipulating the cellular Carbon flux for the production of improved varieties with higher productivity. More specifically, our objective was to modulate the activity of a key regulatory enzyme, the mitochondrial Pyruvate Dehydrogenase Kinase (mt PDHK), for higher Carbon metabolism and subsequent increase in seed weight and Harvest Index, using gene editing technology.

In our initial approach, TALE nucleases (TALEN) would be delivered to haploid embryos using Cell Penetrating Peptide (CPPs) technology. However, our efforts to apply this approach to wheat originally developed in triticale did not reach satisfying results and a more traditional biolistic approach was instead undertaken. By mid-project, our work focused entirely on the bombardment of scutella (diploid embryos) with TALENs, leading to the insertion of single nucleotide mutations at the expected target site of the mt PDHK gene.

This first success in editing the wheat mt PDHK gene in embryos represented a key finding that the TALEN technology undertaken here was indeed a valid approach to reach our goal.

The next step was the regeneration of green plantlets from embryos, which represented another major challenge. To that end, a large part of the project was devoted to establishing an efficient genetic transformation pipeline for wheat, which included the testing of various chemicals to enhanced tissue culture response for increased production of plantlets. A breakthrough came in the last third of the project, with the discovery of a class of molecules that have the ability to significantly increase somatic embryogenesis (SE) to levels required in a high throughput crop improvement platform. This novel, chemical-based (but phytohormone-independent), approach resulted, through the induction of SE-related genes, the significant enhancement of embryogenesis in a non-GMO fashion.

Having established a reliable wheat transformation pipeline, we were finally able to produce satisfying amounts of green plantlets from TALEN-bombarded scutella. Screening of the plantlets produced was performed by Next Generation Sequencing to detect the mutations introduced by TALE nucleases.  Altogether, ~ 100 green plantlets were produced and screened but to this date, none have shown evidence of induced mutations at the target site. Because of the low mutation rate obtained at the embryo level, more individual lines will have to be screened for the detection of such rare mutation events. However, in a parallel study based on the CRISPR technology, we were able to produce, for the first time, a wheat line mutated for the mt PDHK gene by gene editing.  This line is being characterized at the molecular and biochemical levels before being assessed for potential yield increases.

In conclusion, several significant breakthroughs were reached during this 4 year project, the most important one being the establishment of a solid working wheat transformation platform that can be applied, through our proprietary SE chemical enhancer technology, to any other crop of interest to breeders and farmers. In our view, this milestone alone represents a major -and key- step forward in the development of gene editing technology applied to wheat and other crop improvement.