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 ...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.展开更多
基金We thank the financial support from the USDA National Institute of Food and Agriculture SCRI(grant no.2015-70016-23027)the Florida Citrus Development Foundation(2016-001)+1 种基金the Genetically Modified Organisms Breeding Major Projects of China(2014ZX0801008B-001)The Connecticut-Storrs Agriculture Experimental Station,the Priority Academic Program Development of Jiangsu Higher Education Institutions and the Innovative Research Project of JAAS(ZX-17-2006)also contributed financially to some experiments presented in this manuscript.
文摘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.
