Abstract
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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Funding
This research was supported the National Natural Science Foundation of China (Grant no. 31800567), the National Key Program on Transgenic Research (Grant no. 2018ZX08020002), the Innovation and Capacity Building Fund in BAAFS (Grant nos. KJCX20170203 and KJCX20180204), the Beijing Natural Science Foundation (Grant no. 6192007), and the National Key R&D Program of China (Grant no. 2016YFD0600104).
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LPD and JHW designed the research. JHW and HZW supervised the experiments. LPD and YJC conducted the experiments. LPD and YM analyzed the results. LPD and JHW drafted the manuscript. JHW and HZW modified the manuscript. All authors reviewed the manuscript.
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Ding, L., Chen, Y., Ma, Y. et al. Effective reduction in chimeric mutants of poplar trees produced by CRISPR/Cas9 through a second round of shoot regeneration. Plant Biotechnol Rep 14, 549–558 (2020). https://doi.org/10.1007/s11816-020-00629-2
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DOI: https://doi.org/10.1007/s11816-020-00629-2