Interdisciplinary Graduate Program in BIT Medical Convergence, Kangwon National University, Chuncheon 24341, Korea.
Department of Biological Sciences, Kangwon National University, Chuncheon 24341, Korea.
Int J Mol Sci. 2021 Apr 10;22(8):3921. doi: 10.3390/ijms22083921.
Peppers ( L.) are the most widespread and cultivated species of Solanaceae in subtropical and temperate countries. These vegetables are economically attractive worldwide. Although whole-genome sequences of peppers and genome-editing tools are currently available, the precision editing of peppers is still in its infancy because of the lack of a stable pepper transformation method. Here, we employed three strains-AGL1, EHA101, and GV3101-to investigate which strain could be used for pepper transformation. Hot pepper CM334 and bell pepper Dempsey were chosen in this study. GV3101 induced the highest number of calli in cv. Dempsey. All three strains generated similar numbers of calli for cv. CM334. We optimized a suitable concentration of phosphinothricin (PPT) to select a CRISPR/Cas9 binary vector (pBAtC) for both pepper types. Finally, we screened transformed calli for PPT resistance (1 and 5 mg/L PPT for cv. CM334 and Dempsey, respectively). These selected calli showed different indel frequencies from the non-transformed calli. However, the primary indel pattern was consistent with a 1-bp deletion at the target locus of the gene (). These results demonstrate the different sensitivity between cv. CM334 and Dempsey to -mediated callus induction, and a differential selection pressure of PPT via pBAtC binary vector.
辣椒（L.）是茄科植物中在亚热带和温带国家分布最广泛、栽培最多的物种。这些蔬菜在全球范围内具有很高的经济吸引力。尽管目前已经有辣椒的全基因组序列和基因组编辑工具，但由于缺乏稳定的辣椒转化方法，辣椒的精确编辑仍处于起步阶段。在这里，我们使用了三种菌株（AGL1、EHA101 和 GV3101）来研究哪种菌株可用于辣椒转化。本研究选择了辣辣椒 CM334 和甜椒 Dempsey。GV3101 在 Dempsey 品种中诱导出最多的愈伤组织。三种菌株在 CM334 品种中产生的愈伤组织数量相似。我们优化了适宜的膦丝菌素（PPT）浓度，为两种辣椒类型选择了 CRISPR/Cas9 双元载体（pBAtC）。最后，我们筛选了对 PPT 有抗性的转化愈伤组织（CM334 和 Dempsey 的 PPT 浓度分别为 1 和 5mg/L）。这些筛选出的愈伤组织与未转化的愈伤组织的插入缺失频率不同。然而，主要的插入缺失模式与基因（）靶位点的 1bp 缺失一致。这些结果表明，CM334 和 Dempsey 对介导的愈伤组织诱导的敏感性不同，以及 pBAtC 双元载体通过 PPT 产生的差异选择压力。
