Marker-Free Genetic Transformation System Developed for Major Goji Berry Cultivars

Researchers have established an efficient, visually trackable and marker-removable genetic transformation system for Lycium barbarum, the goji berry plant valued for its health-promoting bioactive compounds. Despite decades of effort, regeneration and genetic transformation of this species have remained difficult, limiting genetic research and molecular breeding. The new work, published in BMC Plant Biology, addresses a long-standing gap: no previous transformation system for the species combined high efficiency, broad cultivar applicability, convenient visual screening and the ability to remove non-plant-derived marker elements for biosafety.

The team used the cultivar Zhongxuan-1 as its primary material and constructed a binary vector named pNULPGE708. The vector carries the RUBY visual reporter marker together with the NPT II selectable marker inside a Cre/loxP site-specific recombination cassette driven by the safener-inducible maize ZmIn2-2 promoter. Because RUBY produces a visible, non-invasive signal, the researchers could optimize Agrobacterium tumefaciens strains and antibiotic selection regimes without destructive sampling. They also systematically evaluated explant types, plant growth regulators and culture media for regeneration.

Explant choice proved decisive. Root explants showed a 3- to 5-fold higher capacity for adventitious bud formation than leaf or stem explants. Supplementation of the regeneration medium with 4.31 micromolar melatonin nearly doubled adventitious bud production, raising the yield to 2.69 buds per root explant. Among the Agrobacterium strains tested, GV3101 outperformed AGL1, EHA105 and LBA4404, delivering an average of 0.60 PCR-positive transgenic plants per root explant in Zhongxuan-1.

The system also transferred successfully to other major cultivars. Ningqi-1, Ningqi-5, Ningqi-7 and Ningqi-10 produced 0.38, 0.11, 0.27 and 0.16 PCR-positive transgenic plants per root explant, respectively, all exceeding previously reported values for the species. These figures are averages per explant rather than overall transformation rates, but they represent, according to the authors, the highest efficiencies reported for these cultivars to date.

To generate marker-free plants, the team exploited the inducible recombination system. Treatment with acetanilide induced ZmIn2-2-driven Cre expression, activating Cre/loxP-mediated excision of the marker cassette. Re-differentiation from transgenic roots on medium containing 25 milligrams per liter acetanilide produced the highest average yield, 0.38 marker-excised transgenic plants per re-differentiated root explant, markedly higher than that obtained by direct shoot induction. The authors conclude that the system provides a robust technical foundation for genetic research and molecular breeding in L. barbarum, combining visual tracking of transformed tissue with the option of removing non-plant-derived marker elements.

Subject of Research: Development of an efficient marker-free genetic transformation system for Lycium barbarum cultivars

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

Article References: An, Y., Zhang, H., Lu, G., Yong, J., Chen, J., Lang, S., Liang, W., & Chen, R. (2026). Establishment of an efficient and marker-free transformation system for major cultivars of Lycium barbarum L.. BMC Plant Biology. https://doi.org/10.1186/s12870-026-10275-5

Image Credits: AI Generated

DOI: 10.1186/s12870-026-10275-5

Keywords: Lycium barbarum, goji berry, genetic transformation, Agrobacterium tumefaciens, marker-free, Cre/loxP, RUBY reporter, plant regeneration, melatonin, molecular breeding, Establishment, efficient