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2017 ASHS Annual Conference

Production and High-throughput Screening of Non-transgenic Mutant Plants Resulting from Agrobacterium-mediated Transient CRISPR/Cas9 Expression

Wednesday, September 20, 2017: 3:15 PM
Kohala 3 (Hilton Waikoloa Village)
Yi Li, University of Connecticut, Storrs, CT
Longzheng Chen, Jiangsu Academy of Agricultural Sciences, Nanjing, China
Wei Li, University of Connecticut, Storrs, CT
Lorenzo Katin-Grazzini, University of Connecticut, Storrs, CT
Jing Ding, Nanjing Agricultural University, Nanjing, China
Richard McAvoy, University of Connecticut, Storrs, CT
Fred Gmitter, University of Florida, Lake Alfred, FL
Zhanao Deng, Ph.D., Professor, University of Florida, Wimauma, FL
Audio File Recorded Presentation
Developing CRISPR/Cas9-mediated non-transgenic mutants in asexually propagated perennial crop plants is challenging but highly desirable. We report the development of an Agrobacterium-mediated transient CRISPR/Cas9 gene expression system to create targeted mutations without stable incorporation of T-DNA into the host plant genomes. Additionally, we have developed a rapid, cost-effective, and high-throughput mutant screening protocol based on the novel use of Illumina sequencing followed by high resolution melting (HRM) analysis. Using tobacco as a model species, and the phytoene desaturase (PDS) gene as a target, we obtained pds mutant shoots representing 2.6% of the total number of regenerated shoots, without the use of antibiotic selection during callus or shoot regeneration. Among these pds plants, 17% were confirmed to be non-transgenic. Due to the versatility of CRISPR/Cas9 and Agrobacterium-mediated transient transformation, as well as the ease of plant regeneration from leaf, shoot, or root explants, this method should be applicable to many economically important horticultural plant species.
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