Abstract
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.
Highlights
Transgenic technologies provide powerful tools for crop editing is used in sexually propagated annual crop plants, improvement
Primer sets 1, 2, and 3 were used for PCR analysis to determine the presence of T-DNA fragments stably integrated into the tobacco genome. c The fourth exon of the tobacco phytoene desaturase (PDS) gene was selected as the target site for the single-guide RNA (sgRNA). d, e Histochemical staining of GUS activity in tobacco leaf discs (d) without or (e) with timentin to suppress Agrobacterium growth. d GUS activity in tobacco leaf discs 2–6 days following infection by Agrobacterium. e Reduction in GUS activity in tobacco explants transferred to timentin-containing media, to suppress Agrobacterium growth, for an additional 5 days following the initial 2–6 day co-incubation
To circumvent the limited regeneration potential when using CRISPR ribonucleoproteins to produce non-transgenic mutants, we report a method for using Agrobacterium to transiently express the Cas[9] and sgRNA genes in plant cells, using tobacco as a model plant and PDS as a model target gene
Summary
Transgenic technologies provide powerful tools for crop editing is used in sexually propagated annual crop plants, improvement. The application of these tech- transgenes (Cas[9], sgRNA, and so on) can be eliminated nologies has been hampered by public apprehension from host genomes following sexual reproduction and toward potential food safety and gene flow concerns, screening of segregating populations This segregation of resulting from the presence and/or expression of trans- CRISPR/Cas[9] transgenes from mutations of interest can genes[1,2]. To circumvent the limited regeneration potential when using CRISPR ribonucleoproteins to produce non-transgenic mutants, we report a method for using Agrobacterium to transiently express the Cas[9] and sgRNA genes in plant cells, using tobacco as a model plant and PDS as a model target gene. We demonstrate that the combination of Agrobacteriummediated transient CRISPR/Cas[9] expression with a highly efficient screening protocol makes it possible to efficiently obtain non-transgenic mutant plants, a method that should be applicable to heterozygous perennial crop species
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