CRISPR/Cas9 and Anther Culture for Precision Double Haploid Line Production in Controlled Environments

Abstract

The development of mapping populations and quantitative trait loci (QTL) analysis face constraints, in crops exhibiting male sterility and self‐incompatibility under field conditions. Addressing these challenges requires the integration of advanced techniques, including the temporal alteration or excision of centromere histone H3 (CENH3) protein and the use of gene editing tools such as MATRILINEAL (MTL) knockout. Specifically, this can be achieved through Cas9/gRNA‐mediated mutagenesis or Cas9/gRNA‐driven promoter expression systems. These technologies offer efficient means to advance mapping populations and QTL analysis in male sterile and self‐incompatible crops within controlled ecosystems. The doubled haploid (DH) mapping population, traditionally requiring 3 years of generation time via anther culture method, can now be expedited to 2–3 years of generation time using gene editing techniques within controlled environmental systems. Notably, DH mapping populations can be efficiently generated in various crops, including rice, wheat, maize, barley and oats by leveraging gene editing tools. Among these tools, the novel approach of CENH3 protein temporal alteration/excision emerges as highly efficient compared to MTL knockout using Cas9/gRNA‐mediated mutation or Cas9/gRNA promoter expression. However, further investigation is warranted to optimise the regeneration of double haploid populations and enhance QTL analysis in male sterile and self‐incompatible crops under controlled systems.