An Efficient and Streamlined System for In Vitro Regeneration and Genetic Transformation of Paper Mulberry (Broussonetia papyrifera)

In the present study, we developed an efficient and reproducible protocol for in vitro regeneration and <i>Agrobacterium tumefaciens</i>-mediated genetic transformation of <i>Broussonetia papyrifera</i> (L.) L'Hér. ex Vent. (paper mulberry) using leaf explants from a hybrid genotype. First, we optimized surface sterilization of leaf explants. Treatment with 0.6% (<i>w</i>/<i>v</i>) sodium hypochlorite for 8 min, followed by three rinses with sterile water and blotting on sterile filter paper, yielded a 33.60% explant survival rate and reduced contamination to 35.84%. Second, we refined the co-cultivation step for transformation using <i>A. tumefaciens</i> strain EHA105 carrying pCAMBIA1300-35S-eGFP. Leaf discs were infected for 20 min and co-cultured for 2 days on co-cultivation medium overlaid with sterile filter paper, which limited the overgrowth of <i>A. tumefaciens</i>. After co-cultivation, explants were transferred sequentially to callus induction, shoot induction, shoot multiplication, and rooting media supplemented with 250 mg·L^-1 cefotaxime and 200 mg·L^-1 Timentin, as well as 5.0 mg·L^-1 hygromycin at a concentration that completely suppressed regeneration of non-transformed explants. Meanwhile, after transfer to the callus induction medium, eGFP fluorescence was detected in resistant calli as an initial screening for transformants. The integration and expression of the transgene were further confirmed by PCR and quantitative reverse transcription PCR (qRT-PCR) after the resistant calli developed into plantlets. Collectively, this streamlined protocol provides a practical platform for functional genomics and genetic improvement of <i>B. papyrifera</i>.