Abstract
Key message
Here, we developed a reliable protocol for the fast and efficient gene-edited Anliu sweet orange plants production. The application of in vitro shoot grafting technology significantly reduced the growth cycle of transgenic seedlings, and the survival rate of cleft grafting was more than 90%. In addition, the mutation efficiency of the grafted geneedited sweet orange was significantly improved by short-term heat stress treatments. Thus, the combination strategy of grafting and heat stress treatments provided a reference for the fast and efficient multiplex gene editing of sweet orange.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Belide S, Hac L, Singh SP, Green AG, Wood CC (2011) Agrobacterium-mediated transformation of safflower and the efficient recovery of transgenic plants via grafting. Plant Methods 7(1):12–12
de Oliveira MLP, Febres VJ, Costa MGC, Moore GA, Otoni WC (2009) High-efficiency Agrobacterium-mediated transformation of citrus via sonication and vacuum infiltration. Plant Cell Rep 28(3):387. https://doi.org/10.1007/s00299-008-0646-2
Huang XE, Wang YC, Xu J, Wang N (2020) Development of multiplex genome editing toolkits for citrus with high efficacy in biallelic and homozygous mutations. Plant Mol Biol 104(3):297–307. https://doi.org/10.1007/s11103-020-01043-6
Jia HG, Wang N (2014) Targeted genome editing of sweet orange using Cas9/sgRNA. Plos One 9:e93806. https://doi.org/10.1371/journal.pone.0093806
Jinek M, Chylinski K, Fonfara I, Hauer M, Doudna JA, Charpentier E (2012) A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science 337(6096):816–821. https://doi.org/10.1126/science.1225829
LeBlanc C, Zhang F, Mendez J, Lozano Y, Chatpar K, Irish VF, Jacob Y (2018) Increased efficiency of targeted mutagenesis by CRISPR/Cas9 in plants using heat stress. Plant J 93(2):377–386. https://doi.org/10.1111/tpj.13782
Li HJ, Liu NY, Shi DQ, Liu J, Yang WC (2010) YAO is a nucleolar WD40-repeat protein critical for embryogenesis and gametogenesis in Arabidopsis. BMC Plant Biol 10(1):169. https://doi.org/10.1186/1471-2229-10-169
Wang ZP, Wang SB, Li DW, Zhang Q, Li L, Zhong CH, Liu YF, Huang HW (2018) Optimized paired-sgRNA/Cas9 cloning and expression cassette triggers high-efficiency multiplex genome editing in kiwifruit. Plant Biotechnol J 16(8):1424–1433
Xie KB, Minkenberg B, Yang YN (2015) Boosting CRISPR/Cas9 multiplex editing capability with the endogenous tRNA-processing system. Proc Natl Acad Sci USA 112(11):3570–3575. https://doi.org/10.1073/pnas.1420294112
Zhang F, LeBlanc C, Irish VF, Jacob Y (2017) Rapid and efficient CRISPR/Cas9 gene editing in citrus using the YAO promoter. Plant Cell Rep 36(12):1883–1887. https://doi.org/10.1007/s00299-017-2202-4
Acknowledgements
We are grateful to Dr. Qi Xie from Institute of Genetics and Development, Chinese Academy of Sciences for providing the YAO promoter-driven CRISPR/Cas9 vector and Kabin Xie from College of Plant Sciences and Technology, Huazhong Agricultural University for the guidance of the vectors construction. This work was supported by the National Key Research and Development Program of China (NO. 2018YFD1000101), National Natural Science Foundation of China (NOs. 31925034 and 31872052), and the Fundamental Research Funds for the Central Universities.
Author information
Authors and Affiliations
Contributions
X.T., Q.X. and X.D. conceived and designed the experiments, X.T., S.C., H.Y. and X.Z. conducted the experiments. X.T. and S.C. analyzed data. X.T. wrote the manuscript. Q.X. and F.Z. directed the study and revised the manuscript. All authors read and approved the manuscript.
Corresponding author
Ethics declarations
Conflict of interest
The authors declare no conflict of interest.
Additional information
Communicated by Neal Stewart.
Publisher's Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Supplementary Information
Below is the link to the electronic supplementary material.
Rights and permissions
About this article
Cite this article
Tang, X., Chen, S., Yu, H. et al. Development of a gRNA–tRNA array of CRISPR/Cas9 in combination with grafting technique to improve gene-editing efficiency of sweet orange. Plant Cell Rep 40, 2453–2456 (2021). https://doi.org/10.1007/s00299-021-02781-7
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00299-021-02781-7