B/BE/18/V8

The maize plants have been edited using the CRISPR/Cas9 system resulting in the mutation of either the ALT gene (that plays a role in the repair of DNA damage) or the NGAL2 gene (a transcription factor that has been shown to negatively affect the expression of the PLA1 gene coding for a cytochrome P450 mono-oxygenase enzyme). In both cases one extra DNA base pair has been introduced into the target genes resulting in the knock-out of these genes by means of a frameshift mutation. The plants have thereby become a good biosensor for the type of DNA damage that is caused by different forms of abiotic stress such as drought, heat, or environmental pollution. By knocking out NGAL2 gene the levels of PLA1 may go up resulting in altered growth characteristics, which are phenotypically shown by resulting in a significant larger leaf size. The CRISPR/Cas9 gene cassette, necessary to induce the desired mutation, was introduced into maize plant by means of Agrobacterium tumefaciens mediated transformation. Transformants containing the gene cassette and the desired mutations were selected, and by means of conventional crossing with wild-type plants the CRISPR/Cas9 gene cassette was removed by selecting T1 plants that only contained the desired mutation, but no longer contained the gene cassette (null-segregants). The plants in the field trial therefore do not contain foreign genetic material. They only contain the desired frameshift mutation.