Genetic Transformation Of Foxtail Millet Mediated By Agrobacterium Based On PDS-CRISPR/Cas9 System

Foxtail millet (Setaria italica (L.) Beauv.) is being developed into a kind of model plant for C4 gramineous crops. However, the progress in foxtail millet gene function reseach is slow. The main reason is the low efficiency of genetic transformation. In order to resolve this problem, this experiment aimed to establish an efficient genetic transformation system. In this experiment, the PDS gene in foxtail millet was taken as target gene to constract PDS-CRISPR/Cas vectors. In whole experiment, PDS-CRISPR/Cas9 vectors were taken as main vectors to do the callus transformation and spike dipping experiments to seek for the highly efficient genetic transformation methods for foxtail millet.1. In this experiment, blasting cDNA sequence of PDS gene and PDS gene sequence in corn and genome sequence of foxtail millet respectively, two highly homologous pds genes in foxtail millet were obtained. Choosing three exons of each homologous gene as the target sites, in total,6 pds-CRISPR/Cas9 vectors were constructed successfully.2. In this experiment, using Gugul seed to induce callus and agrobacterium tumefaciens AGLI which contained 6 pds-CRISPR/Cas9 vectors to do the callus transformation. In whole experiment,5 rounds of callus transformation have been completed succeefully and in total,1376 calli have been transformated. Furthermore, the regenerate shoots transformated by each pds-CRISPR/Cas9 vector have been got. Paving the way for further testing of the regenerate plantlets.3. In this experiment, the early flower foxtail millet young spick was chosen as the material for spike dipping and 6 PDS-CRISPR/Cas9 vectors were taken as main vectors to do the spike dipping experiments. In whole experiment,3 rounds of spike dipping have been completed succeefully and in total,74 foxtail millet plants have been transformated. Laying the foundation for further testing of the mature seeds.In this experiment,6 pds-CRISPR/Cas9 vectors were constructed successfully and opening the way for applying CRISPR/Cas9 system to foxtail millet. Due to the limited time, the final experimental results do not be tested but 5 rounds of callus transformation and 3 rounds of spike dipping have been completed succeefully, which laying the firm foundations for further testing experimental results and improving the transformation method.