Haploid induction in allotetraploid tobacco using DMPs mutation

dmp1dmp2dmp3 mutants created by CRISPR/Cas9 could trigger maternal haploids in the allotetraploid model plant Nicotiana tabacum L. Double haploid (DH) technology is becoming increasingly important because it can significantly accelerate the breeding process. Haploid induction plays a fundamental role in the production of DH lines. Haploid induction has been realized and applied in diploid plants using DMP genes. However, it has yet to be elucidated whether haploid induction could be established in polyploid plants. In the current study, three homologues of the DMP genes (NtDMP1, 2, and 3) were identified in the allotetraploid plant Nicotiana tabacum, and the encoded proteins localized in the endoplasmic reticulum. Loss-of-function mutations in all three genes triggered maternal haploids with an induction rate of 1.52-1.75%. Compared with wild-type tobacco, the created haploid inducer exhibited differences in pollen vigor and seed germination rate. Furthermore, to rapidly and easily screen haploids, a visible haploid identification system was established based on a powdery mildew resistance phenotype. Findings from this study lay the foundation for the potential application of haploid inducers in allotetraploid plants such as tobacco.