Knockout of the bread wheat CER9/SUD1 gene using CRISPR/Cas technology

Abstract

Global warming has underscored the imperative of drought tolerance as a paramount trait in bread wheat. Augmenting the cuticular wax layer on leaves and stems stands as one viable approach to enhancing plant drought tolerance. A pivotal discovery surfaced by identifying a mutation within the CER9 gene of Arabidopsis thaliana, amplifying the cuticular wax and consequently mitigating water loss, thereby fortifying drought resilience. Notably, analogous genes, termed SUD1, have been annotated in bread wheat, albeit lacking in-depth scrutiny regarding their phenotypic manifestations under the drought. Hence, our study aimed to employ CRISPR/Cas technology to knockout the CER9/SUD1 gene in bread wheat. For this, five guide RNAs were meticulously chosen and merged into a singular vector. Delivery of the CRISPR/Cas components were arranged through Agrobacterium tumefaciens, utilized for transforming immature embryos of two agricultural spring bread wheat varieties: Taya and Sigma. Among the 13 transgenic plants procured, four manifested positivity for the reporter gene GFP and Cas9 gene. Notably, substantial deletions ranging from 284 bp to 398 bp within the CER9/SUD1 gene were discerned in these plants. Additionally, two of the edited plants exhibited an absence of CER9/SUD1 transcripts, while the other two displayed a noteworthy 5.4-fold reduction in CER9/SUD1 gene expression compared to the wild type. Intriguingly, the genome-edited plants of the T1 generation showcased enhanced growth compared to the wild type under both standard and drought conditions.