Rapid Genetic Stabilisation of Transgenic and Edited Wheat Through Transformation of Immature Haploid Embryos

Wheat is one of the globally important food crops, but its genetically modified application lags behind other staple crops due to its complex genome and low transformation efficiency. Normally, traditional transgenic and gene-edited wheat requires multiple selfing generations for obtaining homozygous lines, which delayed the development of genetically modified wheat varieties. In this study, we crossed a haploid inducer line with purple embryos (HIPE) as a male parent with a wheat cultivar Fielder, and then chose the haploid immature embryos without purple colour to be transformed with the Agrobacterium cells carrying the expression vectors with GUS, RUBY, and Cas9 and sgRNAs for targeting TaWaxy for generating both transgenic and gene editing wheat plants. Consequently, 139 haploid transgenic wheat plants were obtained with transformation efficiencies ranging from 66.7% to 78.4% according to molecular analysis, histochemical staining assay and colour observation. After chromosome doubling treatment to the haploid transgenic plantlets, homozygous transgenic and gene-edited wheat lines were produced in T₀ generation. Genetic analysis of T₁ generation plants confirmed the stable inheritance of the transgenes and edited alleles without segregation. Finally, we established, for the first time, an efficient genetic transformation system based on the use of wheat haploid immature embryos, enabling direct acquisition of homozygous transgenic and gene-edited wheat in T₀ generation. This approach can significantly shorten the wheat transgenic and gene-edited breeding cycle and will provide a novel strategy for the genetic improvement of other polyploid crops.