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Exploring the Agrobacterium-mediated transformation with CRISPR/Cas9 in cucumber (Cucumis sativus L.)

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Abstract

Backgrounds

The narrow genetic basis of cucumber makes breeding of this species difficult. CRISPR/Cas9 system is  characteristic of  simple design, low cost and  high efficiency, which has opened a new path for cucumber functional genetics and the development of cucumber mocular breeding. However, the immature genetic transformation system is the main limiting factor for applying this technology in cucumber.

Methods and Results

In this study, a Histochemical β-glucuronidase (GUS) assay was used to analyze the effect of various parameters, including slight scratch of explants, pre-culture time, acetosyringone (AS) concentration, infection time in Agrobacterium solution, and co-culture period on the transformation efficiency. The results showed that the explants slightly scratched after cutting, pre-cultured for 1 day, Agrobacterium bacterial solution containing AS, and 20 min length of infection could significantly increase the GUS staining rate of explants. On this basis, two sequences with high specificity (sgRNA-1 and sgRNA-2) targeted different loci of gene CsGCN5 were designed. The corresponding vectors Cas9-sgRNA-1 and Cas9-sgRNA-2 were constructed and transformed using the above-optimized cucumber genetic transformation system, and three and two PCR positive lines were obtained from 210 and 207 explants, respectively. No sequence mutation at target loci of CsGCN5 was detected in the Cas9-sgRNA-1 transformed three PCR positive lines. However, one mutant line with targeted homozygous change was recognized from the Cas9-sgRNA-2 transformed two PCR positive lines.

Conclusion

In this study, 2.4‰ of total explants had directed mutation in the CsGCN5 gene. The results in the present study would be beneficial to further optimize and improve the efficiency of the genetic transformation of cucumber.

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Acknowledgements

Work in YL’s lab was supported by the National Natural Science Foundation of China (31772300 and 31471891) and the Shaanxi Province`s Major research and Development Projects (2019TSLNY01-04). Work in PC’s lab and DH’s lab were both supported by the National Natural Science Foundation of China (31860557).

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Author notes

  1. Ziyao Zhao and Yaguang Qi have contributed equally to the study.

Authors and Affiliations

  1. College of Horticulture, Northwest A&F University, Yangling, 712100, Shaanxi, China

    Ziyao Zhao, Yaguang Qi, Zhimin Yang, Liyu Cheng, Rahat Sharif & Yuhong Li

  2. College of Horticulture, Shanxi Agricultural University, Taigu, 030801, Shanxi, China

    Ziyao Zhao

  3. College of Horticulture and Plant Protection, Yangzhou University, Yangzhou, 225009, Jiangsu, China

    Rahat Sharif

  4. Key Laboratory of Ministry of Education for Genetics, Breeding and Multiple Utilization of Crops, Center of Legume Crop Genetics and Systems Biology/College of Agriculture, Oil Crops Research Institute, Fujian Agriculture and Forestry University (FAFU), Fuzhou, China

    Ali Raza

  5. College of Life Science, Northwest A&F University, Yangling, 712100, Shaanxi, China

    Peng Chen

  6. Vegetable Research Institute, Gansu Academy of Agricultural Sciences, Lanzhou, 730070, Gansu, China

    Dong Hou

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  1. Ziyao Zhao
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  2. Yaguang Qi
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Contributions

ZZ and YQ performed the research, prepared a draft of the manuscript. HZ, RS, AR, and LC participated in the research. PC and DH provided technical help. YL designed the experiments, supervised this study. All authors have read and approved the manuscript.

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Correspondence to Yuhong Li.

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Zhao, Z., Qi, Y., Yang, Z. et al. Exploring the Agrobacterium-mediated transformation with CRISPR/Cas9 in cucumber (Cucumis sativus L.). Mol Biol Rep 49, 11481–11490 (2022). https://doi.org/10.1007/s11033-022-07558-z

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