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Agroinfiltration-based transient genome editing for targeting phytoene desaturase gene in kinnow mandarin (C. reticulata Blanco)

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Abstract

Citrus reticulata Blanco also known as kinnow mandarin is a widely grown horticultural crop in Punjab. CRISPR/Cas9 technology is being widely used for generation of varieties with increased resilience towards abiotic and biotic stresses as well as improved horticultural traits. Xanthomonas citri subsp. citri (Xcc)-mediated Agroinfiltration offers a fast and transgene-free method for the delivery of CRISPR/Cas9 constructs for systemic introduction into plants for functional genomics and expression studies. The technology is currently unexplored in kinnow mandarin. This study is aimed at establishing an efficient method of Cas9 delivery for transient knockout of PDS (phytoene desaturase) gene in kinnow mandarin. The construct pKO-119-PDS N-Cas9/sgRNA:PDS1 carrying sgRNA and Cas9 enzyme was delivered into the dorsal surface of young leaves of kinnow mandarin. The leaves showed albino patches at the point of injection within 60 h. Two surfactants (Triton-X and Silwet™) were used to ease the Agroinfiltration process which resulted in variation in the expression of vector. The Sanger’s analysis of the treated plants showed a substitution within the sgRNA region which resulted in change in amino acid from proline to serine. The protocol provides a feasible and an efficient method for genome editing in C. reticulata which could be helpful in future studies aimed at genome editing as well as genetic transformation.

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Acknowledgements

The authors thank Dr. Prashant Mohanpuria, School of Agricultural Biotechnology, Punjab Agricultural University, for providing pKO-119 plasmid procured from Tokushima University, Japan.

Funding

The work is supported by the Department of Biotechnology under the Centre of Excellence Project entitled, ‘Development and Integration of Advanced Genomic Technologies for Targeted Breeding’ CSS-27 (PC-6372).

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  1. School of Agricultural Biotechnology, Punjab Agricultural University, Ludhiana, 141004, India

    Pooja Manchanda, Harleen Kaur, Faishal Khan, Gurupkar S. Sidhu, Parveen Chhuneja & Navtej S. Bains

  2. Department of Plant Pathology, Punjab Agricultural University, Ludhiana, 141004, India

    Mandeep S. Hunjan

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Contributions

PM designed the experiment, arranged the plant material and laboratory facilities, guided FK in conducting the research work and wrote the manuscript. HK conducted data analysis and wrote the manuscript. GSS provided kinnow plants for carrying out Agroinfiltration experiments. MSH provided the Xcc inoculum for carrying out the experiment. PC edited the manuscript. NSB was the mentor of PM and provided the idea for transient genetic transformation in kinnow. All authors reviewed the manuscript.

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Correspondence to Pooja Manchanda.

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Manchanda, P., Kaur, H., Khan, F. et al. Agroinfiltration-based transient genome editing for targeting phytoene desaturase gene in kinnow mandarin (C. reticulata Blanco). Mol Biotechnol (2023). https://doi.org/10.1007/s12033-023-00980-z

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