The legume cowpea (Vigna unguiculata L.) is extensively grown in sub-Saharan Africa. Cowpea, like many legumes has proved recalcitrant to plant transformation. A rapid transient leaf assay was developed for testing gene expression and editing constructs prior to stable cowpea transformation, to accelerate cowpea and legume crop improvement. Attempts to develop a transient protoplast system for cowpea were unsuccessful. Leaflets from plants 3–4 weeks post-germination were age selected to establish a rapid Agrobacterium (Agro) infiltration-mediated transient system for efficacy testing of gene expression and CRISPR/Cas9 gene editing constructs. In planta, Agro-infiltration of leaflets with fluorescent expression constructs, resulted in necrosis. By contrast, Agro-infiltration of detached leaflets with an Arabidopsis (At) ubiquitin3 promoter:ZsGreen construct, followed by culture on solid nutrient medium resulted in fluorescence in over 48% of leaf cells. Expression efficiency was leaf age-dependent. Three cowpea meiosis genes were identified for CRISPR/Cas9 gene-editing, with the forward aim of meiosis-knock out for asexual seed induction in cowpea. Constructs were designed and tested containing candidate gene-specific guide RNAs, expressed using either the cowpea or Arabidopsis U6 promoters with Cas9 expression directed by either the Arabidopsis 40S ribosomal protein or parsley ubiquitin4-2 promoters. Leaflets were infiltrated with test gene-editing constructs and analytical methods developed to identify gene-specific mutations. A construct that produced mutations predicted to induce functional knockout of in the VuSPO11-1 meiosis gene was tested for efficacy in primary transgenic cowpea plants using a previously established stable transformation protocol. Vuspo11-1 mutants were identified, that cytologically phenocopied spo11-1 mutants previously characterized in Arabidopsis, and rice. Importantly, a biallelic male and female sterile mutant was identified in primary transgenics, exhibiting the expected defects in 100% of examined male and female meiocytes. The transient, detached cowpea leaf assay, and supporting analytical methods developed, provide a rapid and reproducible means for testing gene expression constructs, and constructs for inducing mutagenesis in genes involved in both vegetative and reproductive developmental programs. The method and tested editing constructs and components have potential application for a range of crop legumes.

中文翻译：

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用于测试豇豆 (Vigna unguiculata [L.] Walp.) 中瞬时基因表达和基因编辑的分离叶试验。

豆科豇豆 (Vigna unguiculata L.) 在撒哈拉以南非洲广泛种植。与许多豆类一样，豇豆对植物转化具有抗拒性。开发了一种快速瞬时叶片测定法，用于在稳定的豇豆转化之前测试基因表达和编辑构建体，以加速豇豆和豆类作物的改良。为豇豆开发瞬时原生质体系统的尝试没有成功。选择发芽后 3-4 周的植物传单，以建立一个快速农杆菌 (Agro) 浸润介导的瞬态系统，用于基因表达和 CRISPR/Cas9 基因编辑构建体的功效测试。在植物中，带有荧光表达构建体的小叶的农杆菌浸润导致坏死。相比之下，带有拟南芥 (At) 泛素 3 启动子的分离小叶的农业浸润：ZsGreen 构建体，然后在固体营养培养基上培养导致超过 48% 的叶细胞出现荧光。表达效率与叶龄有关。为 CRISPR/Cas9 基因编辑鉴定了三个豇豆减数分裂基因，其远期目标是敲除豇豆的无性种子诱导。设计并测试了包含候选基因特异性指导 RNA 的构建体，使用豇豆或拟南芥 U6 启动子表达，Cas9 表达由拟南芥 40S 核糖体蛋白或欧芹泛素 4-2 启动子指导。传单中渗入了测试基因编辑结构和为识别基因特异性突变而开发的分析方法。使用先前建立的稳定转化方案，在初级转基因豇豆植物中测试了产生预测会诱导 VuSPO11-1 减数分裂基因功能性敲除的突变的构建体在初级转基因豇豆植物中的功效。确定了 Vuspo11-1 突变体，其在细胞学上对 spo11-1 突变体进行了先前在拟南芥和水稻中的表征。重要的是，在初级转基因中发现了一个双等位基因的雄性和雌性不育突变体，在 100% 的检查的雄性和雌性母细胞中表现出预期的缺陷。所开发的瞬时、分离的豇豆叶测定和支持的分析方法为测试基因表达构建体和在涉及营养和生殖发育程序的基因中诱导诱变的构建体提供了一种快速且可重复的方法。