Establishment of an efficient and marker-free transformation system for major cultivars of Lycium barbarum L.

Lycium barbarum is a traditional medicinal and edible plant valued for its health-promoting bioactive compounds. However, despite decades of effort, its regeneration and genetic transformation remain challenging, limiting its genetic research and molecular breeding. In particular, a transformation system combining high efficiency, broad cultivar applicability, convenient visual screening, and the ability to remove non-plant-derived marker elements for biosafety has not yet been established for this species. We used cv. Zhongxuan-1 as the primary material and constructed the binary vector pNULPGE708, which carries the RUBY visual reporter marker and the NPT II selectable marker within a Cre/loxP site-specific recombination system driven by a safener-inducible maize ZmIn2-2 promoter. We evaluated explant types, plant growth regulators and culture media for regeneration, and exploited the visible and non-invasive RUBY to optimize Agrobacterium tumefaciens strains and antibiotic selection regimes for transformation. Root explants exhibited a 3- to 5-fold higher capacity for adventitious bud formation than leaf or stem explants. Supplementation with 4.31 µM melatonin nearly doubled adventitious buds to 2.69 per root explant. Among the tested A. tumefaciens strains, GV3101 outperformed AGL1, EHA105, and LBA4404, with an average of 0.60 PCR-positive transgenic plants per root explant. The system also proved applicable to other major cultivars, including Ningqi-1, Ningqi-5, Ningqi-7, and Ningqi-10, which produced 0.38, 0.11, 0.27, and 0.16 PCR-positive transgenic plants per root explant, respectively, all exceeding previously reported values. To generate marker-free transgenic plants, acetanilide was used to induce ZmIn2-2 -driven Cre expression and activated Cre/loxP-mediated excision. Re-differentiation from transgenic roots on medium containing 25 mg/L acetanilide resulted the highest average of 0.38 marker-excised transgenic plants per re-differentiated root explant, markedly higher than that obtained by direct shoot induction. This study establishes an efficient, visually trackable, and marker-removable transformation system broadly applicable across major cultivars, achieving up to 0.60 PCR-positive transgenic plants per root explant and 0.38 marker-excised transgenic plants per re-differentiated root explant, thereby providing a robust technical foundation for genetic research and molecular breeding in L. barbarum .