Abstract
Papaya is one of the most important fruits in tropical and subtropical countries. However, genetic improvement has had limited success to date due to time-consuming and complex transformation and regeneration technologies, as well as a lack of reproducible and efficient transient gene expression assays. Here, we report the development of a highly efficient Rhizobium rhizogenes-based in vivo hairy root system for evaluating transgene expression and activity including CRISPR/Cas gene editing reagents in the Vietnamese papaya cultivar Linhan. To optimize the papaya transformation parameters, we introduced the R. rhizogenes strain K599 into papaya hypocotyls at 1-, 5- and 10-mm below the cotyledon nodes by a needle using 5-, 7- and 10-day old seedlings and then monitored the frequency of hairy root formation at 18 days post infection. We found that the age of the seedlings and the distance of the infection site from the cotyledon node were inversely correlated with the efficacy of hairy root induction, being 5-day-old plants and 1-mm distance the best parameters. The etablished protocol was then employed to investigate transformation frequency using the gus reporter gene. Of the tested hairy roots, 47.22% were positive for GUS staining, which indicates a high level of transgene transfer and stability. Finally, we introduced a dual guide RNA CRISPR/Cas9 cassette targeting eukaryotic translation initiation factor isoform 4E (eIF(iso)4E) gene into papaya by R. rhizogenes and then screened for gene editing events by heteroduplex analysis and Sanger sequencing. Our analysis revealed that 50% of induced roots contained the expected mutations in the eIF(iso)4E gene, which makes our system ideal for testing transgene activity prior making stable transgenic papaya lines.
Key message
We developed an efficient procedure for papaya in vivo hairy root induction which may be used to validate transgene expression and accelerate CRISPR/Cas-based genome editing studies in papaya.
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Data Availability
All data generated or analysed during this study are included in this published article [and its supplementary information files].
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Acknowledgements
The authors thank Dr. Van Tuong Nguyen for suggestions and discussion on the manuscript.
Funding
This research was supported by the Institute of Biotechnology – Vietnam Academy of Science and Technology (CLCS08.03/22–22) and the Royal Society International Collaboration Award (IC170320).
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HC and PD conceived and supervised the study. TH, NN, and PD designed the study. AM and MM designed, and sequence verified the CRISPR/Cas9 target sites. NL designed the CRISPR/Cas vector. TH and NN performed the experiments. TH and NN wrote the manuscript. LN, TB, ND, AM, and PD assisted in writing the manuscript. NN and PD analyzed the data. NP, AM, HC, and PD revised and proofread the manuscript. All authors contributed to the article and approved the submitted version.
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Communicated by Sergio J. Ochatt.
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Hoang, T.H.T., Nguyen, N.H., Nguyen, L.T. et al. Developing a robust in vivo hairy root system for assessing transgene expression and genome editing efficiency in papaya. Plant Cell Tiss Organ Cult 152, 661–667 (2023). https://doi.org/10.1007/s11240-022-02421-2
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DOI: https://doi.org/10.1007/s11240-022-02421-2