DipTRANS: an improved method for in planta transformation and genome engineering in Nicotiana benthamiana

Plant transformation remains constrained by labor-intensive tissue culture. Our previous work showed that direct delivery of developmental regulators (DRs) can induce de novo meristems on plants, offering a promising transformation approach. In this resource article, we introduced DipTRANS (Direct in planta Transformation), an optimized, soil-based heritable transformation platform for Nicotiana benthamiana that bypasses sterile culture entirely. DipTRANS is built on DR-induced de novo meristem formation. After optimizing parameters, including regulator combinations, Agrobacterium strain, and infiltration density, DipTRANS yielded transformation efficiencies to 46.7%. Developmental abnormalities associated with regulator expression are resolved through cutting-based propagation and virus-induced transgene excision, enabling recovery of fertile, transgenic progeny. Furthermore, DipTRANS supports tissue culture-free, transgene-free iterative genome modification via virus-induced genome editing. Overall, DipTRANS enables the generation of transgenic plants within 30 days and engineered progeny within 90 days. This methodology provides a rapid, versatile platform and a blueprint for extending direct in planta transformation to other plant species.