A robust genome-editing method for wild plant species Nicotiana attenuata
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Plant Biotechnology Reports https://doi.org/10.1007/s11816-020-00634-5
ORIGINAL ARTICLE
A robust genome‑editing method for wild plant species Nicotiana attenuata Moonyoung Kang1 · Hyomin Ahn1 · Eva Rothe2 · Ian T. Baldwin2 · Sang‑Gyu Kim1 Received: 15 April 2020 / Accepted: 14 June 2020 © Korean Society for Plant Biotechnology 2020
Abstract CRISPR genome-editing techniques theoretically enable us to edit any genes in any plants. However, plant tissue culture is required for generating targeted mutants in plants, except in some model plant species such as Arabidopsis thaliana. To modify ecologically or agronomically important traits in plants using the CRISPR system, a robust plant regeneration method and efficient genome-editing tools must be developed. This study shows the entire process of genome editing and the regeneration process for wild tobacco, Nicotiana attenuata. We delivered T-DNA harboring Streptococcus pyogenes Cas9 (SpCas9) and guide RNA (gRNA) via Agrobacterium-mediated transformation into the hypocotyl cells of the wild tobacco and regenerated gene-edited plants. The efficacy of genome editing was measured in N. attenuata protoplasts in which SpCas9 and gRNA were transiently expressed. Light intensity (476.66–627.00 μW/cm 2, 20.69–52.21 μE) was optimized to enhance the emergence of plant shoots during callus induction, and the core step of dealing with plant tissues was recorded. In addition, we found that wounding the bottom part of mature plants was critical for root regeneration. By tracking mutation patterns and efficiency at each regeneration step, we found that the mutation was induced early on in the tissue culture process and maintained throughout the regeneration process. Genome-editing techniques have opened the way to study the function of the genes in all plants, and this study will provide guidelines for editing a gene in the plant of interest. Keywords CRISPR · Plant tissue culture · Regeneration · Wild tobacco · Ecological model species
Introduction Gene silencing mediated by RNA interference (RNAi) has been commonly used to study the physiological and ecological function of genes in plants (Ruiz et al. 1998; Skibbe et al. 2008; Liu et al. 2015). The RNAi technique is especially powerful for studying both the function of gene families with high sequence similarity and also the function of lethal genes. However, the RNAi technique has potential disadvantages: it induces non-specific gene silencing, and Electronic supplementary material The online version of this article (https://doi.org/10.1007/s11816-020-00634-5) contains supplementary material, which is available to authorized users. * Sang‑Gyu Kim [email protected] 1
Department of Biological Sciences, KAIST, Daejeon 34141, South Korea
Department of Molecular Ecology, Max Planck Institute for Chemical Ecology, Hans‑Knöll‑Str. 8, 07745 Jena, Germany
2
sometimes produces obscure phenotypes if target genes are incompletely silenced (Boutros and Ahringer 2008; Rajeevkumar et al. 2015). The CRISPR (clustered regularly
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