GE-TEA Rice lines were generated by site-directed mutagenesis using CRISPR/Cas9, delivered via co-cultivation of in vitro embryogenic calli of Oryza sativa japonica with Agrobacterium tumefaciens. Nine independent lines were produced using the cultivars ‘Telemaco’, ‘Vialone Nano’, ‘Limnee Tommaso’, and ‘Pacifico’.

The editing process targets five genes, Pi21, HMA1, HMA2, Gn1a and Os04g0621500. Pi21, HMA1, HMA2, and Os04g0621500 are involved in the susceptibility to the pathogen Pyricularia oryzae (syn. Magnaporthe oryzae), causal agent of the rice blast disease. Gn1a mitigates production losses under stress conditions.

Following genome editing in the T0 generation, the transgene encoding hygromycin resistance, Cas9, and gRNAs were segregated out in the T1 generation. Absence of the transgenes was determined using PCR and genome sequencing. The deletions at the intended target sites were confirmed by PCR and Sanger sequencing. Homozygous plants lacking the transgene and expected deletions or insertions in the coding sequences of the target genes were selected.

Source: BCH Italy (SNIF B/IT/25/01 and SNIF B/IT/24/01); machine translation of the documents)