The CRISPR/Cas9 technology, renowned for its ability to induce precise genetic alterations in various crop species, has encountered challenges in its application to grain legume crops such as pigeonpea and groundnut. Despite attempts at gene editing in groundnut, the low rates of transformation and editing have impeded its widespread adoption in producing genetically modified plants. This study seeks to establish an effective CRISPR/Cas9 system in pigeonpea and groundnut through Agrobacterium-mediated transformation, with a focus on targeting the phytoene desaturase (PDS) gene. The PDS gene is pivotal in carotenoid biosynthesis, and its disruption leads to albino phenotypes and dwarfism. Two constructs (one each for pigeonpea and groundnut) were developed for the PDS gene, and transformation was carried out using different explants (leaf petiolar tissue for pigeonpea and cotyledonary nodes for groundnut). By adjusting the composition of the growth media and refining Agrobacterium infection techniques, transformation efficiencies of 15.2% in pigeonpea and 20% in groundnut were achieved. Mutation in PDS resulted in albino phenotype, with editing efficiencies ranging from 4 to 6%. Sequence analysis uncovered a nucleotide deletion (A) in pigeonpea and an A insertion in groundnut, leading to a premature stop codon and, thereby, an albino phenotype. This research offers a significant foundation for the swift assessment and enhancement of CRISPR/Cas9-based genome editing technologies in legume crops.

CRISPR/Cas9技术以其在多种作物物种中诱导精确遗传改变的能力而闻名，但在木豆和花生等豆科作物的应用中遇到了挑战。尽管尝试对花生进行基因编辑，但转化和编辑率较低，阻碍了其在生产转基因植物中的广泛采用。本研究旨在通过农杆菌介导的转化，在木豆和花生中建立有效的 CRISPR/Cas9 系统，重点是针对八氢番茄红素去饱和酶( PDS ) 基因。PDS基因在类胡萝卜素生物合成中至关重要，其破坏会导致白化病表型和侏儒症。针对PDS基因开发了两种构建体（木豆和花生各一种），并使用不同的外植体（木豆的叶柄组织和花生的子叶节）进行转化。通过调整培养基成分和完善农杆菌侵染技术，木豆转化效率达到15.2%，花生转化效率达到20%。PDS突变导致白化表型，编辑效率范围为 4% 至 6%。序列分析发现木豆中存在核苷酸缺失 (A)，花生中存在 A 插入，导致终止密码子过早出现，从而产生白化表型。这项研究为豆类作物中基于 CRISPR/Cas9 的基因组编辑技术的快速评估和增强奠定了重要基础。