Department of Soil and Crop Sciences, Texas A&M University, College Station, TX, United States of America.
PLoS One. 2023 Apr 5;18(4):e0283837. doi: 10.1371/journal.pone.0283837. eCollection 2023.
Cowpea (Vigna unguiculata) is a legume staple widely grown across Sub-Saharan Africa and other tropical and sub-tropical regions. Considering projected climate change and global population increases, cowpea's adaptation to hot climates, resistance to drought, and nitrogen-fixing capabilities make it an especially attractive crop for facing future challenges. Despite these beneficial traits, efficient varietal improvement is challenging in cowpea due to its recalcitrance to transformation and long regeneration times. Transient gene expression assays can provide solutions to alleviate these issues as they allow researchers to test gene editing constructs before investing in the time and resource- intensive process of transformation. In this study, we developed an improved cowpea protoplast isolation protocol, a transient protoplast assay, and an agroinfiltration assay to be used for initial testing and validation of gene editing constructs and for gene expression studies. To test these protocols, we assessed the efficacy of a CRISPR-Cas9 construct containing four multiplexed single-guide RNA (sgRNA) sequences using polyethylene glycol (PEG)-mediated transformation and agroinfiltration with phytoene desaturase (PDS) as the target gene. Sanger sequencing of DNA from transformed protoplasts and agroinfiltrated cowpea leaves revealed several large deletions in the target sequences. The protoplast system and agroinfiltration protocol developed in this study provide versatile tools to test gene editing components before initiating plant transformation, thus improving the chance of using active sgRNAs and attaining the desired edits and target phenotype.
豇豆（Vigna unguiculata）是一种在撒哈拉以南非洲和其他热带及亚热带地区广泛种植的豆类作物。考虑到预计的气候变化和全球人口增长，豇豆对炎热气候的适应能力、对干旱的抵抗力和固氮能力使其成为应对未来挑战的一种极具吸引力的作物。尽管具有这些有益的特性，但由于其转化的顽固性和再生时间长，豇豆的高效品种改良具有挑战性。瞬时基因表达分析可以提供解决方案来缓解这些问题，因为它们允许研究人员在投入时间和资源密集型的转化过程之前，测试基因编辑构建体。在这项研究中，我们开发了一种改良的豇豆原生质体分离方案、瞬时原生质体测定和农杆菌浸润测定，用于初步测试和验证基因编辑构建体以及基因表达研究。为了测试这些方案，我们评估了含有四个多重单引导 RNA (sgRNA) 序列的 CRISPR-Cas9 构建体的功效，该构建体使用聚乙二醇 (PEG) 介导的转化和以脱番茄红素酶 (PDS) 为靶基因的农杆菌浸润。转化原生质体和农杆菌浸润豇豆叶片的 DNA 的 Sanger 测序显示靶序列中存在几个大片段缺失。本研究中开发的原生质体系统和农杆菌浸润方案为在启动植物转化之前测试基因编辑组件提供了多功能工具，从而提高了使用活性 sgRNA 并获得所需编辑和目标表型的机会。
