Abstract

Banana is an important food crop worldwide. However, as banana production is largely affected by pests, diseases, and environmental stresses, there is a need to develop stress-resistant banana varieties. Although modern breeding and conventional genetic engineering techniques exist, they may not provide sufficient precision or speed in introducing desired traits and enhancing banana resilience. Hence, this study focuses on the development of an efficient protoplast isolation and DNA-free CRISPR/Cas9 ribonucleoprotein-mediated protoplast transformation protocols for Berangan cultivar. A total of 1.54 × 10⁷ protoplasts/g FW were isolated from immature male flower buds using an enzymatic mixture of 1% cellulase RS, 1% macerozyme R-10 and 0.15% pectolyase Y-23. Applying 10 min-vacuum infiltrations twice and 0.5 M mannitol significantly increased the number of protoplasts. The isolated protoplasts were transfected with pC-AMBIA1304-GFP and CRISPR/Cas9 ribonucleoprotein complex targeting the stress-related banana Sugar Transport Protein 13 (STP13) using a polyethylene glycol solution. Following 15 minutes of transfection, 76.89% of the treated protoplasts were GFP-positive. DNA sequence analysis confirmed the presence of small deletions (1–3 bp) at the target sites of STP13 with a mutation rate of 4.40–4.90%, indicating that the protocols are suitable for use to modify the genomes of protoplasts of bananas.

中文翻译：

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PEG辅助香蕉原生质体转染系统的建立 Berangan (AAA) 使用 CRISPR/Cas9 核糖核蛋白复合物

摘要

香蕉是全世界重要的粮食作物。然而，由于香蕉生产在很大程度上受到病虫害和环境压力的影响，因此需要开发抗逆香蕉品种。尽管存在现代育种和传统基因工程技术，但它们可能无法提供足够的精度或速度来引入所需性状和增强香蕉的恢复力。因此，本研究的重点是为 Berangan 品种开发有效的原生质体分离和无 DNA CRISPR/Cas9 核糖核蛋白介导的原生质体转化方案。使用1％纤维素酶RS、1％macerozyme R-10和0.15％果胶酶Y-23的酶混合物从未成熟雄性花芽中分离出总共1.54×10 ^{7原生质体/g FW。}应用两次 10 分钟真空渗透和 0.5 M 甘露醇显着增加了原生质体的数量。使用聚乙二醇溶液，将分离的原生质体转染 pC-AMBIA1304-GFP 和 CRISPR/Cas9 核糖核蛋白复合物，靶向应激相关的香蕉糖转运蛋白 13 ( STP13 )。转染 15 分钟后，76.89% 的处理原生质体呈 GFP 阳性。DNA序列分析证实STP13的靶位点存在小缺失（1-3bp），突变率为4.40-4.90%，表明该方案适合用于修饰香蕉原生质体基因组。