CRISPR/Cas9-induced Targeted Mutagenesis and Gene Replacement to Generate Long-shelf Life Tomato Lines

• Published in: Scientific reports Vol. 7; no. 1; pp. 11874 - 9
• Main Authors: Yu, Qing-Hui, Wang, Baike, Li, Ning, Tang, Yaping, Yang, Shengbao, Yang, Tao, Xu, Juan, Guo, Chunmiao, Yan, Peng, Wang, Qiang, Asmutola, Patiguli
• Format: Journal Article
• Language: English
• Published: England Nature Publishing Group 19.09.2017; Nature Publishing Group UK
• Subjects: Agrobacterium tumefaciens - genetics; Agrobacterium tumefaciens - metabolism; CRISPR; CRISPR-Cas Systems; Gene Editing; Gene Targeting; Genes, Plant; Genetic transformation; Genome editing; Genotyping; Insertion; Lycopersicon esculentum - genetics; Lycopersicon esculentum - metabolism; Mutagenesis; Mutagenesis, Site-Directed; Mutation; Plant breeding; Plants, Genetically Modified - genetics; Plants, Genetically Modified - metabolism; Shelf life; Site-directed mutagenesis; T-DNA; Transgenic plants
• ISSN: 2045-2322; 2045-2322
• DOI: 10.1038/s41598-017-12262-1

Quickly and precisely gain genetically enhanced breeding elites with value-adding performance traits is desired by the crop breeders all the time. The present of gene editing technologies, especially the CRISPR/Cas9 system with the capacities of efficiency, versatility and multiplexing provides a reasonable expectation towards breeding goals. For exploiting possible application to accelerate the speed of process at breeding by CRISPR/Cas9 technology, in this study, the Agrobacterium tumefaciens-mediated CRISPR/Cas9 system transformation method was used for obtaining tomato ALC gene mutagenesis and replacement, in absence and presence of the homologous repair template. The average mutation frequency (72.73%) and low replacement efficiency (7.69%) were achieved in T transgenic plants respectively. None of homozygous mutation was detected in T transgenic plants, but one plant carry the heterozygous genes (Cas9/*-ALC/alc) was stably transmitted to T generations for segregation and genotyping. Finally, the desired alc homozygous mutants without T-DNA insertion (*/*-alc/alc) in T generations were acquired and further confirmed by genotype and phenotype characterization, with highlight of excellent storage performance, thus the recessive homozygous breeding elites with the character of long-shelf life were generated. Our results support that CRISPR/Cas9-induced gene replacement via HDR provides a valuable method for breeding elite innovation in tomato.