Abstract
Broccoli, a Brassica crop, is an important vegetative crop because of its nutritional components and beneficial phytochemicals. Glucoraphanin (GR), a major glucosinolate (GSL) in broccoli, is converted by hydrolyzation of the endogenous enzyme, myrosinase, into sulforaphane (SR), which protects the body against a variety of chronic diseases. Despite their economic importance, biotechnological approaches for increasing GR content in Brassica species are still limited. The main objective of this study was to develop a GR-rich broccoli cultivar using the CRISPR/Cas9-mediated DNA-free genome-editing technique. It is considered that MYB28 is one of the key genes involved in the accumulation of GSL levels in broccoli. Furthermore, with increased GSL levels by introgression of MYB28 from wild species, B. villosa showed a 9 bp deletion in exon 3, leading to one amino acid substitution and the deletion of three amino acids. Therefore, we considered the 9 bp deletion to be the most significant change in GR-rich broccoli and conducted Cas9 protein and single-guide RNA transfection into broccoli protoplasts for editing the flanking sequence of the 9 bp deleted MYB28 gene. Finally, increased GR content was observed in broccoli regenerated from protoplasts with specifically edited MYB28.
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Acknowledgements
This work was supported by the grants: the Bio-industry Technology Development Program (Grant No. 117043-3) of iPET (Korea Institute of Planning and Evaluation for Technology in Food, Agriculture, and Forestry), the cooperative Research Program for Agriculture Science and Technology Development (Project No. PJ01487201) of Rural Development Administration, Republic of Korea, and the Basic Science Research Program of the National Research Foundation (NRF) funded by the Republic of Korea’s Ministry of Education (Grant No. 2020R1I1A1A01072914). We thank Jeong Hwan Lee and Young Jae Yun (Jeonbuk National University) for helpful discussion and technical assistance.
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Kim, YC., Ahn, W.S., Cha, A. et al. Development of glucoraphanin-rich broccoli (Brassica oleracea var. italica) by CRISPR/Cas9-mediated DNA-free BolMYB28 editing. Plant Biotechnol Rep 16, 123–132 (2022). https://doi.org/10.1007/s11816-021-00732-y
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DOI: https://doi.org/10.1007/s11816-021-00732-y