Comparison of protoplast isolation methods.

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）是一种豆科主粮作物，广泛种植于撒哈拉以南非洲及其他热带、亚热带地区。鉴于预估的气候变化与全球人口增长态势，豇豆对高温环境的适应性、抗旱性及固氮能力，使其成为应对未来粮食挑战的极具潜力的作物。尽管具备上述优良性状，但豇豆的遗传转化顽拗性与漫长的再生周期，使得其高效品种改良颇具挑战。瞬时基因表达分析可有效缓解这些难题，允许研究人员在投入耗时耗力的遗传转化流程之前，先行测试基因编辑构建体。本研究开发了优化后的豇豆原生质体分离方案、瞬时原生质体检测体系及农杆菌浸润检测体系，可用于基因编辑构建体的初步测试与验证，以及基因表达研究。为验证上述实验方案，我们以八氢番茄红素脱氢酶（phytoene desaturase, PDS）为靶基因，分别采用聚乙二醇（polyethylene glycol, PEG）介导的转化法与农杆菌浸润法，对包含4条多重单向导RNA（single-guide RNA, sgRNA）序列的CRISPR-Cas9构建体的编辑效能进行了评估。对转化后的原生质体及经农杆菌浸润的豇豆叶片的DNA进行桑格测序，结果显示靶序列中存在多处大片段缺失。本研究开发的原生质体检测体系与农杆菌浸润方案，可在启动植物遗传转化前用于测试基因编辑元件，从而提升使用活性sgRNA、获得预期编辑效果及目标表型的概率。