Timed Self-Excision of PlWUS-IPT Resolves the Transformation Efficiency-Phenotype Trade- off in Tobacco

Overexpression of morphogenic regulators such as WUS-IPT dramatically enhances plant regeneration and transformation efficiency, yet it invariably causes severe developmental defects that prevent the recovery of normal plant. Here, using tobacco ( Nicotiana tabacum ) leaf explants, we developed an estradiol-inducible Cre/loxP self-excision system to exploit the transient regenerative power of WUS-IPT while avoiding its long-term pleiotropic effects. Under standard conditions, the PIWUS-IPT (PIWI) construct increased transformation efficiency in leaf discs by 4.7-fold relative to the empty vector control (79.2% vs. 16.7%). Remarkably, PIWI also enabled hormone-free callus and shoot regeneration, achieving a transformation efficiency of 54.3%. By systematically optimizing the excision timing, we discovered that applying 10 µM estradiol at 3 days post-cocultivation—rather than at 0 or 7 days—fully rescued the aberrant phenotypes and, unexpectedly, boosted overall transformation efficiency to 105% (a 1.35-fold improvement over the non-excised control), yielding completely normal, non-chimeric plants. Furthermore, deployment of the PIWI helper system in PDS gene knockout experiments substantially increased the recovery of albino edited lines, with sequencing confirming target mutations. Our study establishes a precise temporal strategy for deploying morphogenic regulators, providing a powerful new paradigm for overcoming regeneration bottlenecks and advancing functional genomics and genome editing in recalcitrant plant species.