The canola event 5715 was developed by conventional breeding of two herbicide tolerant mutants.
Brassica napus carries two complete genomes designated "A" and "C". Each genome has an acetohydroxyacid synthase (ahas) gene (ahas1 gene in the C genome and ahas3 in the A genome) coding for an AHAS enzyme.
Event 5715 is a cross between Cibus canola line BnALS-57 (donor of the single nucleotide mutation in the ahas1 gene of the C genome) and the commercially available Clearfield canola variety SP Cougar CL (donor of the single nucleotide mutation in the ahas3 gene of the A genome).
BnALS-57 was selected as a mutant variant of the wild-type parental line BN2. Mutants of BN2 were generated using an oligonucleotide-directed mutagenesis (ODM) approach known as the Rapid Trait Development System™ (RTDS™).
No changes were observed in the promoter regions or locations that would affect endogenous gene expression. This was confirmed by sequencing a 2500-base pair (bp) region upstream of the AHAS1 gene which was determined to be identical with GenBank accession Z11524. The developer hypothesized that the single nucleotide mutation was the result of a spontaneous somaclonal variation that occurred during the tissue culture process, and not due to the specific oligonucleotide used in the RTDS protocol.
Being a crossing product of BnALS-57 and SP Cougar CL, 5715 possesses a specific single nucleotide mutation in the ahas1 and ahas3 gene, respectively. Both the ahas1 and ahas3 genes encode a protein subunit (i.e., a monomer) of an acetohydroxyacid synthase (ahas) enzyme. The single nucleotide mutation in the subunits of each of the ahas enzymes results in conformational changes in their configuration, reducing the binding affinity of imidazolinone and sulfonylurea herbicides, thus conferring herbicide tolerance to the canola plant.
DNA sequencing was used to confirm homozygosity of the single nucleotide mutation in both the ahas1 and ahas3 genes. Furthermore, plants from an advanced breeding generation of Cibus canola event 5715 demonstrated consistent imidazolinone and sulfonylurea herbicide tolerance, phenotypically confirming the stable inheritance of the trait.