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.

中文翻译：

---

通过第二轮芽再生有效减少CRISPR / Cas9产生的杨树嵌合突变体

簇状调控间隔的短回文重复/ CRISPR-相关蛋白9（CRISPR / Cas9）系统已被用于编辑许多植物物种的基因组，包括多年生木本杨树。但是，嵌合体通常存在于初级转基因植物中。对于多年生木本植物，例如杨树，由于其植物寿命长和结实率低，很难通过自花授粉获得纯合突变体。在这项研究中，我们报告了一种有效的方法，可通过CRISPR / Cas9降低杨树嵌合突变体的频率，并使用叶子作为外植体进行第二轮芽再生。PdbPDS1被用作靶基因，只有一个纯合的PdbPDS1从15个原代转基因小植株中获得了突变体，并通过PCR产物的表型和DNA序列进行了验证。这表明在T0代中，大多数主要的转基因苗是嵌合体。在第二轮由CRISPR / Cas9产生的嵌合突变体的芽再生之后，分别有或没有选择卡那霉素的纯合突变体分别约为27.0％或19.1％。结果表明，第二次再生可以高频率产生纯合突变体芽，而卡那霉素的选择可以增加纯合突变体芽的频率。