Improved efficiency of genetic transformation for the development of elite germplasm using CRISPR/Cas12i3 in maize

Genetic transformation and gene-editing technologies have driven progress in molecular design breeding. Jing 724, an elite founder inbred line from the X heterotic group in maize, was previously recalcitrant to genetic transformation owing to genotype-dependent restrictions. Here, we established a stable and efficient genetic transformation system for Jing 724 using the morphogenic genes Baby boom (Bbm) and Wuschel (Wus2) and the selection marker 6-phosphomannose isomerase (PMI). Under identical Bbm/Wus2-regulation conditions, the efficiencies of callus induction, regeneration, and transformation were significantly higher (30 %) with PMI (mannose) selection than with bar (bialaphos) selection. By optimizing callus induction and regeneration time, we reduced the transformation period for Jing 724 from 90 days to 60 days and increased the transformation efficiency by 20 %. We then combined this transformation system with the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated nuclease 12i3 (Cas12i3) system to edit the Sugary1 (Su1) and Waxy (Wx) genes, thereby developing elite germplasms with common sweet, waxy, and combined sweet-waxy traits in the Jing 724 background. We obtained mutation efficiencies of 29.41 % (homozygous), 41.18 % (heterozygous), and 35.29 % (simultaneous). Additionally, concentrations of sucrose, water-soluble polysaccharides, and starch in the endosperms of T₁ homozygous kernels were confirmed to differ significantly from those in wild-type kernels. Thus, we have established a highly efficient molecular breeding system that integrates genetic transformation, gene editing, and germplasm development.