Open AccessA method for the production and expedient screening of CRISPR/Cas9-mediated non-transgenic mutant plants
Author(s) -
Longzheng Chen,
Wei Li,
Lorenzo KatinGrazzini,
Jing Ding,
Xianbin Gu,
Yanjun Li,
Tingting Gu,
Ren Wang,
Xinchun Lin,
Zhiming Deng,
Richard McAvoy,
Frederick G. Gmitter,
Zhanao Deng,
Yunde Zhao,
Yang Li
Publication year - 2018
Publication title -
horticulture research
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.947
H-Index - 31
eISSN - 2662-6810
pISSN - 2052-7276
DOI - 10.1038/s41438-018-0023-4
Subject(s) - biology , mutant , transgene , genetically modified crops , cas9 , crispr , genetics , gene , agrobacterium
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.