Developing CRISPR/Cas9-mediated non-transgenic mutants in asexually propagated perennial crop plants is challenging but highly desirable. Here, we report a highly useful method using an Agrobacterium-mediated transient CRISPR/Cas9 gene expression system to create non-transgenic mutant plants without the need for sexual segregation. We have also developed a rapid, cost-effective, and high-throughput mutant screening protocol based on Illumina sequencing followed by high-resolution melting (HRM) analysis. Using tetraploid tobacco as a model species and the phytoene desaturase (PDS) gene as a target, we successfully created and expediently identified mutant plants, which were verified as tetra-allelic mutants. We produced pds mutant shoots at a rate of 47.5% from tobacco leaf explants, without the use of antibiotic selection. Among these pds plants, 17.2% were confirmed to be non-transgenic, for an overall non-transgenic mutation rate of 8.2%. Our method is reliable and effective in creating non-transgenic mutant plants without the need to segregate out transgenes through sexual reproduction. This method should be applicable to many economically important, heterozygous, perennial crop species that are more difficult to regenerate.

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SUMMARY Trifoliate orange (Poncirus trifoliata), a deciduous close relative of evergreen Citrus, has important traits for citrus production, including tolerance/resistance to citrus greening disease (Huanglongbing, HLB) and other major diseases, and cold tolerance. It has been one of the most important rootstocks, and one of the most valuable sources of resistance and tolerance genes for citrus. Here we present a high‐quality, chromosome‐scale genome assembly of P. trifoliata. The 264.9‐Mb assembly contains nine chromosomal pseudomolecules with 25 538 protein‐coding genes, covering 97.2% of the estimated gene space. Comparative analyses of P. trifoliata and nine Citrus genomes revealed 605 species‐specific genes and six rapidly evolving gene families in the P. trifoliata genome. Poncirus trifoliata has evolved specific adaptation in the C‐repeat/DREB binding factor (CBF)‐dependent and CBF‐independent cold signaling pathways to tolerate cold. We identified candidate genes within quantitative trait loci for HLB tolerance, and at the loci for resistance to citrus tristeza virus and citrus nematode. Genetic diversity analysis of Poncirus accessions and Poncirus/Citrus hybrids shows a narrow genetic base in the US germplasm collection, and points to the importance of collecting and preserving more natural genetic variation. Two phenotypically divergent Poncirus accessions are found to be clonally related, supporting a previous conjecture that dwarf Flying Dragon originated as a mutant of a non‐dwarfing type. The high‐quality genome reveals features and evolutionary insights of Poncirus, and it will serve as a valuable resource for genetic, genomic and molecular research and manipulation in citrus.