Pandey Prashant Kumar, Bhowmik Pankaj, Kagale Sateesh
Aquatic and Crop Resource Development, National Research Council Canada, Saskatoon, SK, Canada.
Front Plant Sci. 2022 Sep 8;13:995542. doi: 10.3389/fpls.2022.995542. eCollection 2022.
Field pea is an important pulse crop for its dense nutritional profile and contribution to sustainable agricultural practices. Recently, it has received extensive attention as a potential leading source of plant-based proteins. However, the adoption of peas as a mainstream source of proteins is affected by a relatively moderate protein content, anti-nutritional factors and high levels of off-flavor components that reduce protein quality. Availability of genetic variation for desirable seed quality traits is the foundation for the sustainable development of pea varieties with improved protein content and quality. Mutagenesis has been an important tool in gene functional characterization studies and creating genetic variability for crop breeding. Large-scale mutagenesis of a crop using physical and chemical agents requires diligent selection of the mutagen and optimization of its dose to increase the frequency of mutations. In this study, we present detailed optimized protocols for physical and chemical mutagenesis of pea using gamma irradiation and ethyl methanesulfonate (EMS), respectively. Gamma radiation and EMS titration kill curves were established to identify optimal doses of the two mutagenic agents. Based on germination, survival rate and growth phenotypes, a gamma radiation dose of 225 Gy and EMS concentration of 5 mm were selected as optimal dosages for mutagenesis in field pea. The presented protocol has been modified from previously established mutagenesis protocols in other crop plants. Our results indicate that the optimal mutagen dosage is genotype dependent. CRISPR/Cas-based gene editing provides a precise and rapid method for targeted genetic manipulation in plants. With the recent success of gene editing in pea using CRISPR/Cas, this innovative technology is expected to become an integral component of the gene discovery and crop improvement toolkit in pea. Here, we describe an optimized methods for targeted mutagenesis of pea protoplasts, including mesophyll protoplast extraction, PEG-mediated transformation and gene editing of a LOX gene using CRISPR/Cas system. The general strategies and methods of mutagenesis described here provide an essential resource for mutation breeding and functional genomics studies in pea. These methods also provide a foundation for similar studies in other crops.
豌豆因其丰富的营养成分和对可持续农业实践的贡献，是一种重要的豆类作物。最近，它作为植物性蛋白质的潜在主要来源受到了广泛关注。然而，豌豆作为蛋白质的主流来源受到相对适中的蛋白质含量、抗营养因子以及降低蛋白质质量的高水平异味成分的影响。获得理想种子品质性状的遗传变异是培育蛋白质含量和质量得到改善的豌豆品种可持续发展的基础。诱变一直是基因功能表征研究以及为作物育种创造遗传变异的重要工具。使用物理和化学试剂对作物进行大规模诱变需要精心选择诱变剂并优化其剂量，以提高突变频率。在本研究中，我们分别介绍了使用伽马射线辐照和甲基磺酸乙酯（EMS）对豌豆进行物理和化学诱变的详细优化方案。建立了伽马辐射和EMS滴定致死曲线，以确定这两种诱变剂的最佳剂量。基于发芽率、存活率和生长表型，选择225 Gy的伽马辐射剂量和5 mM的EMS浓度作为豌豆诱变的最佳剂量。所提出的方案是在其他作物先前建立的诱变方案基础上修改而来的。我们的结果表明，最佳诱变剂量取决于基因型。基于CRISPR/Cas的基因编辑为植物中的靶向遗传操作提供了一种精确且快速的方法。随着最近在豌豆中使用CRISPR/Cas进行基因编辑取得成功，这项创新技术有望成为豌豆基因发现和作物改良工具包的一个组成部分。在这里，我们描述了一种用于豌豆原生质体靶向诱变的优化方法，包括叶肉原生质体提取、聚乙二醇介导的转化以及使用CRISPR/Cas系统对一个脂氧合酶（LOX）基因进行基因编辑。这里描述的诱变的一般策略和方法为豌豆的突变育种和功能基因组学研究提供了重要资源。这些方法也为其他作物的类似研究奠定了基础。
