Induction of chromosomal aberrations with ectopic expression of native BABY BOOM1 (GmBBM1) in soybean

Haploid induction is of great importance in the breeding of cross-pollinated crops such as maize, and it also expedites the development of new varieties in a shorter period in self-pollinated crops, i.e. soybean. The success rate of traditional techniques is almost negligible at less than 1%, and their applicability is dependent on external factors in soybean. There is a lack of standardized and reproducible systems, which makes it challenging to adopt the existing systems for haploid plant production. Therefore, there is a high demand for implementing innovative approaches for this crop due to the limitations of conventional methods. The BABY BOOM (BBM) gene, which generated haploidy at high rates (> 80%) when expressed in pearl millet or through ectopic expression in the egg cells of rice, maize, and tobacco, is a novel example with promising potential. In this study, we used the egg cell-specific promoter DD45, which was cloned from Arabidopsis, to ectopically express the native soybean BBM1 (GmBBM1) gene in soybean to observe the response to haploidy induction. Initially, the clone pDD45:GmBBM1 was successfully constructed and confirmed by PCR and Sanger sequencing. The construct was subsequently transformed into soybean via a half-seed approach. The expression of GmBBM1 in both flowers and leaves increased in the T₀ transgenic soybean lines. The T₀ plants and their seeds showed developmental abnormalities described by early senescence and flowering; however, the T₁ plants exhibited normal growth characteristics. The ploidy levels of the T₁ and T₂ plants were determined by flow cytometry and chromosome counting. The flow cytometry histograms revealed haploidy in the T₁ generation; however, further chromosome counting in T₂ plants revealed changes in chromosome number, and aneuploidy, which may be due to spontaneous doubling. This system is especially important in legume crops, as no reports exist on the application of the BBM1 system in soybean. Our study will provide valuable insights for future research and advancing soybean breeding with haploid induction.