PRJNA800116
Highly Efficient Agrobacterium rhizogenes-mediated Genetic Transformation Technology Bypassing Tissue Culture in Citrus
Highly efficient genetic transformation technology is of great significance for the gene function analysis and precision breeding of crops. However, the most commonly used genetic transformation technology mediated by Agrobacterium tumefaciens in plants especially xylophyta is time-consuming and inefficient, which seriously hinders the progress of gene function analysis. In this study, a simple and highly efficient genetic transformation technology mediated by Agrobacterium rhizogenes bypassing tissue culture is described. Only 2~8 weeks were required for the whole workflow, with an average successful transformation ratio of 57% in shoots from adult plants. By using this technology, we successfully transferred plasmids containing gRNA, Cas9, and other exogenous genes into citrus, and achieved genome editing on target loci and overexpressed multiple foreign genes at the same time. In this method, a student could simultaneously conduct genome editing experiment using 10 plasmids targeting different genome positions and finally obtained all the corresponding transformants with target DNA knocked out, indicating that A. rhizogenes-mediated genome editing technology is highly efficient. In addition, A. rhizogenes can be used for direct viral vector inoculation on citrus bypassing the step of viron enrichment in tobacco, which facilitates the experiment operation of virus-induced gene silence (VIGS) and foreign gene expression. In summary, we established a highly efficient genetic transformation technology bypassing tissue culture in citrus, which can be used for genome editing, gene overexpression, and virus-mediated gene function analysis. We anticipate that by removing high cost, heavy workload, long experiment period and other technical obstacles, this genetic transformation technology will be a valuable tool for routine investigation of endogenous and exogenous genes in citrus.