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

ABSTRACT Grain amaranth is a nutritionally rich, stress‐tolerant C 4 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.