TRTech-PGR: Rapid Transformation and Editing in Maize

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Transformation and gene editing are critical procedures for genomic analysis and will be important for agriculture in the coming years to keep pace with the need to feed the world. There are two aspects to the recovery of stable transgenes and gene edits in plants, namely introducing the new genes into plant cells and then the subsequent recovery of the transformed or edited cells. This project will develop a system in corn for placing transgenes at predetermined sites in the genome in a recombination reaction as opposed to the standard procedure of random integration. The system is designed to be able to continue to add new genes at these pre-determined sites with subsequent transformation events. This system will be introduced into a fast-flowering mini-maize variety that can be grown up to 6-7 generations per year. This variety is not only fast developing but also smaller than normal corn and thus can be grown in growth chambers in controlled environments. This variety can be used to induce and edit haploids of maternal plants with only one copy of the genome that can then be doubled into edited diploids. The target lines can be normal corn of any variety, so the editing of any line of corn can be rapid using this technique. If successful, an extremely efficient and extremely rapid technique for both transformation and gene editing of various sorts will be available to the plant biology community. Landing pads that will accept additions mediated by phiC31 integrase will be placed on five different normal chromosomes of maize in two different versions of fast flowering mini-maize. A copy of the landing pad on the B chromosome will allow gene stacking on this supernumerary chromosome that can be used for increasing the copy number of transgenes or removal of the same via the nondisjunction property of the B at the mitosis that produces the two sperm. The phiC31 integrase will be used to place genes at landing pads and recovered using selectable markers and a morphogenic regulator. The selectable marker and morphogenic regulator will be removed using Cre recombinase, retaining the gene cargo. Multiple subsequent additions of genes can be added to the same sites through a reiterative process. Gene stacking at the landing pads will be conducted as a proof of concept to place multiple genes into the genome. Gene editing has become an integral part of genomic analysis so the system will be adapted by converting the stacking lines to haploid inducers. These lines can produce edits in haploid genomes to be doubled to recover the edits or crossed as females to rescue the edits as heterozygotes in the same inbred line. Once a specific haploid inducing editing line is produced for specific gene targets, the stock can be perpetuated and used to edit any line regardless of its transformability and without the need for tissue culture. All project outcomes will be readily accessible by the public. Sequence data will be available through the NCBI SRA/GEO and biological resources either upon request or through long-term repositories such as the Maize Genetic Cooperation Stock Center and Addgene.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.