First Successful Targeted Mutagenesis Using CRISPR/Cas9 in Stably Transformed Grain Amaranth Tissue.

Grain amaranth is a nutritionally rich, stress-tolerant C4 dicot with considerable potential for climate-resilient agriculture; however, efficient and reproducible protocols for stable transformation, regeneration, and CRISPR/Cas9-mediated editing have not yet been established. CRISPR/Cas-based genome editing is a cornerstone technology for accelerating the development of climate-resilient, high-yielding crops. Its effective application depends on robust, stable transformation procedures and CRISPR/Cas systems optimised for the target species. The absence of such tools remains a critical constraint for the genetic improvement of many promising yet underexplored crops. In this study, we edited key genes of the betalain biosynthesis pathway in grain amaranth (Amaranthus hypochondriacus L.) using the CasCADE modular cloning system, thereby demonstrating the feasibility of targeted mutagenesis in an orphan crop. We observed successful edits in up to 49% of transformed calli, resulting in deletions or insertions in the target genes. Our CRISPR/Cas9-mediated editing paves the way for targeted molecular research and breeding of grain amaranth.