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

TLDR

Crop breeders seek rapid, precise genetic enhancement, and CRISPR/Cas9 offers efficient, versatile, multiplexing capabilities that promise to meet these breeding goals. The study aimed to accelerate breeding by using Agrobacterium‑mediated CRISPR/Cas9 to mutate and replace the tomato ALC gene, with and without a homologous repair template. The authors employed Agrobacterium tumefaciens‑mediated CRISPR/Cas9 transformation to generate ALC gene mutations and replacements, testing both with and without a homologous repair template. In T0 transgenic tomatoes, a 72.73 % mutation rate and 7.69 % replacement efficiency were observed, with no homozygous mutants but a heterozygous line transmitted to T1, from which homozygous alc mutants lacking T‑DNA were recovered and displayed superior shelf life, demonstrating the utility of HDR‑mediated CRISPR/Cas9 gene replacement for elite tomato breeding.

Abstract

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 T0 transgenic plants respectively. None of homozygous mutation was detected in T0 transgenic plants, but one plant carry the heterozygous genes (Cas9/*-ALC/alc) was stably transmitted to T1 generations for segregation and genotyping. Finally, the desired alc homozygous mutants without T-DNA insertion (*/*-alc/alc) in T1 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.