Coupling FT-Mediated Speed Breeding and CRISPR/Cas9 for Rapid Trait Improvement in Nicotiana tabacum

The commercial deployment of genome-edited crops is frequently bottlenecked by the extended juvenile phases of perennial species and the complex regulatory landscapes governing plants with integrated exogenous DNA. Speed breeding protocols, which utilize environmental manipulation to accelerate development, have proven effective for annual cereals but often lack efficacy in perennials. In this study, we report a genetic speed breeding system that couples the overexpression of the floral integrator FLOWERING LOCUS T ( FT ) with CRISPR/Cas9-mediated genome editing. Using Nicotiana tabacum as a model for polyploid crops, we demonstrate that constitutive expression of Arabidopsis thaliana FT ( AtFT ) reduces the generation time from 12 weeks to approximately 3 weeks without compromising fertility or seed viability. To leverage this acceleration for trait improvement, we engineered a binary vector co-expressing AtFT and a CRISPR/Cas9 cassette targeting the NtDFR ( dihydroflavonol 4-reductase ) loci. This integrated system induced rapid flowering and simultaneous disruption of anthocyanin biosynthesis, yielding ntdfr mutants with a distinct white-flower phenotype. Importantly, the edited alleles were heritable, while the FT-Cas9 transgene could be segregated out in the next generation, restoring the wild-type photoperiodic response in the edited progeny. This transgenic facilitator approach offers a scalable platform for the rapid introgression of edited traits into recalcitrant crop species, potentially reducing the breeding cycle of perennials from years to months.