Targeted allelic diversification of flowering genes via CRISPR-Cas enables the development of multiple early-flowering soybean lines.

CRISPR-Cas multiplex editing generated a broad range of early-flowering soybean lines, advancing flowering by up to four weeks under controlled long-day conditions and by approximately one to two weeks in a field trial. Soybean is a promising crop for European agriculture, but its cultivation area remains smaller than its potential. A major limitation to broader adoption in Europe is the restricted number of elite lines adapted to local environmental conditions, particularly the photoperiod encountered at higher latitude. At these latitudes, soybean must flower early to complete its growth cycle before the onset of harsh autumn conditions. In this project, we employed a CRISPR-Cas multiplex knockout strategy, to generate allelic diversity in the gene network controlling flowering time, with the aim of developing earlier flowering soybeans lines better suited to European high latitudes. This approach successfully produced earlier flowering varieties, with flowering advances of up to four weeks compared to the controls. Starting from maturity group (MG) I and MG III lines, we developed, within two generations, gene-edited early flowering soybean lines with estimated maturity ranges between MG 000 and MG 0. Greenhouse observations were supported by a field trial, in an MG II environment, where edited lines flowered approximately one to two weeks earlier than their respective parental controls. These results demonstrate that multiplex editing of flowering-time genes can rapidly generate targeted allelic diversity and a broad spectrum of flowering phenotypes within elite soybean backgrounds. These results provide valuable breeding material and a genotype/phenotype resource for subsequent evaluation of flowering-time adaptation across environments.