Institute of Horticulture, Xinjiang Academy of Agricultural Science, Urumqi, 830091, China.
Sci Rep. 2017 Sep 19;7(1):11874. 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.
一直以来，作物育种家都希望能够快速、准确地培育出具有附加值的遗传改良型品种。基因编辑技术的出现，特别是具有高效性、多功能性和多重性的 CRISPR/Cas9 系统，为实现这些育种目标提供了合理的预期。为了探索 CRISPR/Cas9 技术在加速育种进程方面的可能应用，本研究利用农杆菌介导的 CRISPR/Cas9 系统转化方法，在不存在和存在同源修复模板的情况下，对番茄 ALC 基因进行诱变和替换。在 T 代转基因植株中，分别获得了 72.73%的平均突变频率和 7.69%的低替换效率。在 T 代转基因植株中未检测到纯合突变，但有一株携带杂合基因（Cas9/-ALC/alc），其稳定传递到 T 代进行分离和基因分型。最后，在 T 代获得了期望的 alc 纯合突变体，且没有 T-DNA 插入（/*-alc/alc），并通过基因型和表型特征进一步确认，具有出色的贮藏性能，从而产生了具有长货架期特征的隐性纯合型优良品种。我们的研究结果表明，CRISPR/Cas9 诱导的 HDR 基因替换为番茄的优良品种创新提供了一种有价值的方法。
