A method for the production and expedient screening of CRISPR/Cas9-mediated non-transgenic mutant plants

文献类型： 外文期刊

第一作者： Chen, Longzheng

作者： Chen, Longzheng;Li, Wei;Katin-Grazzini, Lorenzo;Gu, Xianbin;Li, Yanjun;Wang, Ren;Lin, Xinchun;McAvoy, Richard J.;Li, Yi;Chen, Longzheng;Ding, Jing;Gu, Tingting;Li, Yi;Ding, Jing;Gu, Tingting;Li, Yi;Lin, Xinchun;Deng, Ziniu;Gmitter, Frederick G., Jr.;Deng, Zhanao;Zhao, Yunde

作者机构：

期刊名称：HORTICULTURE RESEARCH （ 影响因子：6.793； 五年影响因子：6.589 ）

ISSN： 2052-7276

年卷期： 2018 年 5 卷

页码：

收录情况： SCI

摘要： 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.

分类号：

  • 相关文献
作者其他论文 更多>>

微信小程序