CRISPR/Cas9 ribonucleoproteins enable DNA-free genome editing; thus, improving protoplast culture is essential for the efficient development of mutant plants. However, the use of protoplast cultures is limited because a universal method cannot be applied to diverse plants. Solanum lycopersicum ‘Heinz 1706,’ a model cultivar for tomato genome analysis, has not yet been studied for DNA-free genome editing. We optimized the protoplast culture method for the tomato model cultivar ‘Heinz 1706’ using combinations of plant growth regulators (PGRs) and basal media. Isolated protoplasts were cultured in R-Ini medium for cell division and micro-calli proliferation, and then the medium was changed to G-207.3 medium for callus formation. Among different concentrations of 1-naphthaleneacetic acid (NAA) and 6-benzylaminopurine (BAP), the combination of 0.05 mg/L NAA and 0.5 mg/L BAP in the R-Ini medium was observed to be highly efficient for micro-calli development. G-207.3 liquid medium was more efficient for mini-calli formation than the R-Ini liquid medium. For calli formation from the mini-calli, 0.1 mg/L 2,4-dichlorophenoxyacetic acid (2,4-D), 0.05 mg/L NAA, and 0.5 mg/L BAP were used in G-207.3 solid medium. In addition, five single guide RNAs (sgRNAs) were designed to target SlPelo using polyethylene glycol-mediated transfection, thereby developing a tool for tomato yellow leaf curl virus (TYLCV) resistance breeding. sgRNAs and Cas9 complexes were delivered into protoplasts using PEG-mediated transfection, and sgRNA2 resulted in a high mutagenesis efficiency. The results will be valuable for DNA-free genome editing in tomato for the development of new breeding materials.

CRISPR/Cas9核糖核蛋白实现无DNA基因组编辑；因此，改善原生质体培养对于突变植物的有效发育至关重要。然而，原生质体培养的使用受到限制，因为通用方法不能应用于不同的植物。番茄“Heinz 1706”是一种用于番茄基因组分析的模型品种，尚未进行无 DNA 基因组编辑的研究。我们使用植物生长调节剂（PGR）和基础培养基的组合优化了番茄模型品种“Heinz 1706”的原生质体培养方法。分离的原生质体在R-Ini培养基中培养进行细胞分裂和微愈伤组织增殖，然后将培养基更换为G-207.3培养基进行愈伤组织形成。在不同浓度的 1-萘乙酸 (NAA) 和 6-苄氨基嘌呤 (BAP) 中，R-Ini 培养基中 0.05 mg/L NAA 和 0.5 mg/L BAP 的组合对微愈伤组织的发育非常有效。G-207.3 液体培养基对于微型愈伤组织的形成比 R-Ini 液体培养基更有效。对于小型愈伤组织的愈伤组织形成，0.1 mg/L 2,4-二氯苯氧基乙酸 (2,4-D)，G-207.3固体培养基中使用0.05 mg/L NAA和0.5 mg/L BAP。此外，还设计了 5 个单向导 RNA (sgRNA) 来靶向SlPelo利用聚乙二醇介导的转染，从而开发了番茄黄曲叶病毒（TYLCV）抗性育种的工具。使用 PEG 介导的转染将 sgRNA 和 Cas9 复合物递送到原生质体中，并且 sgRNA2 具有很高的诱变效率。这些结果对于番茄的无 DNA 基因组编辑以及新育种材料的开发具有重要价值。