Efficient recovery of transgene-free edited tobacco using the transgene self-elimination system via de novo regeneration

In Agrobacterium-mediated de novo regenerated tobacco, the DLL-driven BARNASE transgene self-elimination system produced 83.72% uniformly marker-negative progeny lineages (36/43) from pEB138.

• Why it matters: Integrated editing constructs are hard to remove in plant genome editing, limiting recovery of transgene-free progeny after Agrobacterium-mediated regeneration.
• What they did: Tobacco leaf-disc transformation used three pU3TR-derived vectors coupling CRISPR/Cas9 to developmentally regulated BARNASE cassettes (DLL, OsRP3, PHAS), with lineage-level screening testing up to 48 T progeny per T line.
• The result: pEB138 showed 81.16% T plants with mutations at one or both NtDFR loci (56/69) and enabled recovery of dfr mutants and NtA622-edited lines, including NtBBL progeny with reduced nicotine and loss of the selectable cassette.