Abstract:

【Objective】 Because cassava（Manihot esculenta Crantz）contains potentially toxic cyanogenic glucosides, its food safety is affected, which leads to the increase of processing costs. It is of great significance to cultivate cassava with low cyanogenic glycosides by biotechnology.【Method】 CRISPR/Cas9 technology was used to edit the α-hydroxynitrile lyase gene MeHNL. This gene encodes α-hydroxynitrile lyase that catalyzes the decomposition of cyanogenic glycosides. The editing target is located on its first exon, and 27 positive plants were obtained through Agrobacterium-mediated stable transformation.【Result】Sequencing analysis indicated successful editing in 26 out of 27 transgenic plants, and a high editing efficiency was achieved by 96.3%. The editing types predominantly comprised base insertions and deletions, alongside minor base substitutions and large fragment deletions. HCN colorimetric kit and HPLC analysis confirmed a significant reduction in hydrocyanic acid and cyanogenic glycosides in the mutant lines. Additionally, transgenic plants demonstrated slender leaves compared to non-transgenic counterparts, implicating MeHNL's impact on plant growth and development.【Conclusion】The cassava germplasm with low cyanide is obtained by CRISPR/Cas9 technology, which provides materials for exploring the study of cyanogenic glycoside metabolism on cassava growth and development.

Key words: cassava, gene editing, HNL, low cyanide, cyanogenic glycosides