International Crops Research Institute for the Semi-Arid Tropics (ICRISAT), Hyderabad, 502324, India.
Funct Integr Genomics. 2024 Mar 13;24(2):57. doi: 10.1007/s10142-024-01336-9.
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 技术以其在各种作物物种中诱导精确基因改变的能力而闻名，但在应用于豇豆和落花生等粮食豆科作物时却遇到了挑战。尽管在落花生中尝试了基因编辑，但转化和编辑的低效率阻碍了其在生产转基因植物中的广泛应用。本研究旨在通过农杆菌介导的转化在豇豆和落花生中建立有效的 CRISPR/Cas9 系统，重点是针对类胡萝卜素生物合成中的脱饱和酶（PDS）基因。PDS 基因在类胡萝卜素生物合成中起着关键作用，其失活会导致白化表型和矮化。针对 PDS 基因分别为豇豆和落花生构建了两个构建体（各一个），并使用不同的外植体（豇豆的叶叶柄组织和落花生的子叶节点）进行转化。通过调整生长培养基的组成和优化农杆菌感染技术，豇豆的转化效率达到了 15.2%，落花生的转化效率达到了 20%。PDS 基因的突变导致白化表型，编辑效率在 4%到 6%之间。序列分析发现豇豆中存在一个核苷酸缺失（A），落花生中存在一个 A 插入，导致提前出现终止密码子，从而产生白化表型。这项研究为快速评估和增强豆科作物基于 CRISPR/Cas9 的基因组编辑技术提供了重要基础。
