We demonstrated also that CsACS2, like CmACS-7, is specifically expressed in carpel primordia of buds determined to develop carpels. However, in the absence of transgenic expression data or targeted mutagenesis approach in cucumber, we were not able to clearly conclude that the M locus in cucumber encodes for CsACS2 gene. Here we report the creation of a mutant collection from monoecious cucumber line under controlled conditions and the set up of a TILLinG platform. We validated the quality of the mutagenesis by screening for induced mutations in 5 genes, mainly involved in sex determination and LY2109761 plant architecture processes, and characterized TILLinG lines harboring induced mutations in the Monoecious sex gene, CsACS2. This work lead to the identification of a missense mutation in CsACS2 resulting in a sexual transition from monoecy to andromonoecy. Based on this, we concluded that that the M gene encodes for CsACS2. This result also demonstrates that TILLinG is an efficient tool for functional validation of gene function in crops recalcitrant to transgenic transformation. Monoecy is characterized by the presence of both male and female flowers on the same plant. In cucumber, this sexual type is controlled by the identity of the alleles at the M locus. In melon,LY2157299 we previously showed that the transition from monoecy to andromonoecy results from a mutation in 1-aminocyclopropane-1-carboxylic acid synthase gene, CmACS-7. To isolate the andromonoecy gene in cucumber, we previously used a candidate gene approach in combination with genetic and biochemical analysis. We demonstrated co-segregation of CsACS2 gene, the cucumber homolog of CmACS-7 in melon, with the M locus in cucumber. However, in the absence of transgenic expression data or targeted mutagenesis approach in cucumber, we were not able to clearly conclude that the M locus in cucumber encodes for CsACS2 gene. As the cucumber mutant collection described above was created from a monoecious line homozygous for the M locus, we took advantage of the cucumber TILLinG platform and screened for induced mutations in the full genomic sequence of CsACS2.