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Establishment of an Agrobacterium-mediated genetic transformation and CRISPR/Cas9-mediated mutagenesis of haploid inducer genes in Pak-choi plants (Brassica rapa ssp. chinensis)

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Abstract

Pak-choi (Brassica rapa ssp. chinensis) is a popular vegetative crop in southern China, East Asia, and Southeast Asia. Owing to the threat of climate change, rapid breeding strategies for vegetable cultivars that are tolerant to abiotic and biotic stresses are required. Thus, the rapid fixation of useful agronomic traits using doubled haploid technology is urgent. The haploid-inducer gene is key to doubled haploidization. Two known CENH3 and pPLAIIγ genes, in which altered or partially deleted forms lead to haploid induction, were selected, and direct editing of Pak-choi CENH3 and pPLAIIγ genes (BcCENH3 and BcpPLAIIγ) was conducted using an Agrobacterium-mediated CRISPR/Cas9 system. First, BcCENH3 and BcpPLAIIγ genes were characterized by analyzing the spatial expression patterns and subcellular localization. The CENH3 expression levels in carpels and pPLAIIγ in various parts of Pak-choi flowers were higher than those of other parts. BcCENH3 and BcpPLAIIγ proteins targeted in the nucleus and plasma membrane, respectively. Whole plants were successfully regenerated from the shoot apical meristem (SAM) regions of Pak-choi seedlings using the optimized procedure and culture conditions. The regeneration results of SAM explants after Agrobacterium-mediated transformation of constructs expressing CRISPR/Cas9 and BcCENH3 or BcpPLAIIγ sgRNAs confirmed four independent BcCENH3-targeted transgenic lines with 2.1%, 1.8%, 1.8%, and 1.7% INDEL frequencies, and three independent BcpPLAIIγ-targeted transgenic lines with 24.5%, 33.7%, and 33.0% INDEL frequencies. Thus, our results suggested the possibility of developing transgenic Pak-choi lines by applying the CRISPR/Cas9 genome editing technology to BcCENH3 and BcpPLAIIγ as two haploid-inducer genes.

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Acknowledgements

We thank E. B. Choi, H. H. Jeong, and M. Chen for their supporting tissue culture experiment. We also thank Dr. H. Ryu for kindly providing research material (a RFP-tagged AtARR2 marker) and Ms. Y. Y. Choi, Center for University-wide Research Facilities (CURF) at Jeonbuk National University, for confocal microscopic observations. This work was supported by grants from the Basic Science Research Program of the National Research Foundation (NRF) (2020R1I1A1A01072914 to Y.-C. Kim and 2019R1A2C1004140 to O. R. Lee) and the Rural Development Administration (RDA) (RS-2024-00322297 to J. H. Lee), Republic of Korea. This work was also supported by research funds of Jeonbuk National University in 2022 (to J. H. Lee) and BK21 FOUR Program by Jeonbuk National University Research Grant (to M. P. Thu).

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  1. Young-Cheon Kim and May Phyo Thu contributed equally to this work.

Authors and Affiliations

  1. Department of Life Sciences, Jeonbuk National University, 567 Baekje-daero, Deokjin-gu, Jeollabuk-do, 54896, Jeonju, Korea

    Young-Cheon Kim, May Phyo Thu, Falguni Maliha Rahman, Young Jae Yun & Jeong Hwan Lee

  2. HOPS Biosciences, 567 Baekje-daero, Deokjin-gu, Jeollabuk-do, 54896, Jeonju, Korea

    Young-Cheon Kim & Jeong Hwan Lee

  3. Department of Applied Plant Science, College of Agriculture and Life Sciences, Chonnam National University, Gwangju, 61186, Korea

    Jin Hoon Jang & Ok Ran Lee

  4. Interdisciplinary Program in IT-Bio Convergence System, Chonnam National University, Gwangju, 61186, Korea

    Jin Hoon Jang & Ok Ran Lee

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Contributions

YCK and JHL conceived and designed the study; MPT performed the majority of research with the help of FMR, YJY, and JHJ; YCK, ORL, and JHL completed the final manuscript; all authors have read and agreed to the published version of the manuscript.

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Correspondence to Jeong Hwan Lee.

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Kim, YC., Thu, M.P., Rahman, F.M. et al. Establishment of an Agrobacterium-mediated genetic transformation and CRISPR/Cas9-mediated mutagenesis of haploid inducer genes in Pak-choi plants (Brassica rapa ssp. chinensis). Plant Biotechnol Rep 18, 263–273 (2024). https://doi.org/10.1007/s11816-024-00898-1

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