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Article|02 Mar 2018|OPEN
A method for the production and expedient screening of CRISPR/Cas9-mediated non-transgenic mutant plants
Longzheng Chen1,2, Wei Li1, Lorenzo Katin-Grazzini1, Jing Ding3, Xianbin Gu1, Yanjun Li1, Tingting Gu3, Ren Wang1, Xinchun Lin1,4, Ziniu Deng5, Richard J. McAvoy1, Frederick G. GmitterJr.6, Zhanao Deng7, Yunde Zhao8 & Yi Li3,1,
1Department of Plant Science and Landscape Architecture, University of Connecticut, Storrs, CT, USA
2Institute of Vegetable Crops, Jiangsu Academy of Agricultural Sciences, Nanjing, China
3College of Horticulture and State Key Laboratory of Crop Genetics and Germplasm Enhancement, Nanjing Agricultural University, Nanjing, China
4State Key Laboratory of Subtropical Silviculture, Zhejiang Agriculture and Forestry University, Zhejiang Hangzhou China
5College of Horticulture, Hunan Agricultural University, Hunan, Changsha, China
6Citrus Research and Education Center, University of Florida, Lake Alfred, FL, USA
7Department of Environmental Horticulture, Gulf Coast Research and Education Center, IFAS, University of Florida, Wimauma, FL, USA
8Section of Cell and Developmental Biology, University of California at San Diego, San Diego, CA 92093, USA

Horticulture Research 5,
Article number: 13 (2018)
doi: 10.1038/hortres.2018.13
Views: 841

Received: 05 Feb 2018
Revised: 05 Feb 2018
Accepted: 06 Feb 2018
Published online: 02 Mar 2018


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.