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/cm², 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.

中文翻译：

---

野生植物烟烟草的一种可靠的基因组编辑方法

从理论上讲，CRISPR基因组编辑技术使我们能够编辑任何植物中的任何基因。然而，除了在一些模型植物物种中，例如拟南芥（Arabidopsis thaliana），在植物中产生靶向突变体需要植物组织培养。为了使用CRISPR系统修饰植物在生态上或农艺上重要的性状，必须开发出可靠的植物再生方法和有效的基因组编辑工具。这项研究显示了全基因组编辑的整个过程以及野生烟草（弱烟）的再生过程。我们提供了带有化脓链球菌Cas9（SpCas9）的T-DNA，并通过农杆菌介导了RNA（gRNA）介导的转化为野生烟草和再生的基因编辑植物的下胚轴细胞。基因组编辑的功效是在N. plea原生质体中测量的，其中SpCas9和gRNA瞬时表达。光强度（476.66–627.00μW/ cm ²（20.69–52.21μE）进行了优化，以增强愈伤组织诱导期间植物嫩芽的出现，并记录了处理植物组织的核心步骤。此外，我们发现，伤害成熟植物的底部对于根再生至关重要。通过跟踪每个再生步骤的突变模式和效率，我们发现该突变在组织培养过程的早期被诱导，并在整个再生过程中得以维持。基因组编辑技术为研究所有植物中基因的功能开辟了道路，这项研究将为编辑目的植物中的基因提供指导。