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Effective reduction in chimeric mutants of poplar trees produced by CRISPR/Cas9 through a second round of shoot regeneration

文献类型： 外文期刊

作者： Ding, Liping 1 ; Chen, Yajuan 1 ; Ma, Yan 2 ; Wang, Hongzhi 1 ; Wei, Jianhua 1 ;

作者机构： 1.Beijing Acad Agr & Forestry Sci, Beijing Key Lab Agr Gene Resource & Biotechnol, Beijing Agrobiotechnol Res Ctr, Beijing 100097, Peoples R China

2.Jinling Inst Technol, Coll Hort & Landscape Architecture, Nanjing 210000, Peoples R China

关键词： CRISPR; Cas9; Chimeric mutants; Homozygous mutants; Second regeneration; Poplar

期刊名称：PLANT BIOTECHNOLOGY REPORTS （ 影响因子：2.01； 五年影响因子：1.907 ）

ISSN： 1863-5466

年卷期： 2020 年 14 卷 5 期

页码：

收录情况： SCI

摘要： The clustered regulatory interspaced short palindromic repeat/CRISPR-associated protein 9 (CRISPR/Cas9) system has been applied to edit the genomes of quite a few plant species, including perennial woody poplar trees. However, chimeras often exist in primary transgenic plants. For perennial woody trees such as poplar trees, it is difficult to obtain homozygous mutants by self-pollination for due to their long vegetative life and low-seed setting rates. In this study, we report an effective approach to reduce the frequency of chimeric mutants of poplar trees by CRISPR/Cas9 with a second round of shoot regeneration using leaves as the explants. PdbPDS1 was used as the target gene, and only one homozygous PdbPDS1 mutant was obtained from 15 primary transgenic plantlets, which was verified by both the phenotype and the DNA sequence of the PCR product. This indicates that the majority of primary transgenic plantlets in the T0 generation were chimeras. After the second round of shoot regeneration of the chimeric mutants generated by CRISPR/Cas9, approximately 27.0% or 19.1% of the regenerated shoots were homozygous mutants with or without kanamycin selection, respectively. The results showed that a second regeneration could produce homozygous mutant shoots at a high frequency and that kanamycin selection could increase the frequency of homozygous mutant shoots.

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