Establishment of an efficient and reliable Agrobacterium-mediated genetic transformation system for the elite
Zilu Qin1, Haiyan Sun1, Jingwei Li1
1State Key Laboratory of Tree Genetics and Breeding, Key Laboratory of Forest Genetics & Biotechnology of Ministry of Education, Co-Innovation Center for Sustainable Forestry in Southern China, Nanjing Forestry University, Nanjing, 210037, China.
Abstract:
'Siyang-1' (Populus×euramericana 'Siyang-1') is a male-sterile elite cultivar widely cultivated in southern China. However, with the expansion of cultivation area, 'Siyang-1' plantations are increasingly threatened by insect pests, especially the fall webworm (Hyphantria cunea), which causes substantial economic and ecological damage. To genetically engineer an insect-resistant cultivar using Bacillus thuringiensis (Bt) toxin genes, an efficient and reliable Agrobacterium tumefaciens-mediated transformation system was established. This was achieved by first developing an in vitro whole-plant regeneration system using petiole explants, which showed the highest adventitious shoot induction on SIM14 medium (0.5 mg·L⁻¹ 6-BA and 0.2 mg·L⁻¹ IAA), followed by effective rooting on RM7 medium (0.5 mg·L⁻¹ IBA). The optimal kanamycin selection pressure was determined to be 50 mg·L⁻¹. Critical transformation parameters were systematically optimized, identifying bacterial concentration (OD₆₀₀=0.6), infection time (20 min), and co-cultivation period (2 days) with strain EHA105 as the most effective combination. Using this optimized protocol, transgenic 'Siyang-1' plants co-expressing two Bt genes, Cry1Ac and Cry3A, were successfully generated. Genomic PCR and semi-quantitative RT-PCR verified transgene integration and expression. Leaf-feeding bioassays demonstrated that these transgenic lines exhibited significantly enhanced resistance against the H. cunea, with leaf area consumption reduced to 5.28%-12.66%, compared to approximately 50% in wild-type plants. Enzyme-Linked Immunosorbent Assay (ELISA) quantification further confirmed the presence of Cry1Ac protein in the transgenic lines, and a positive correlation was observed between Cry1Ac protein level and the reduction in leaf consumption. The combination of insect resistance and male sterility makes these lines a valuable germplasm resource for poplar breeding, with potential advantages for biosafety due to reduced pollen-mediated gene flow. The established transformation platform not only addresses a major technical challenge to genetically improving 'Siyang-1', but also provides a foundational tool for future gene-editing and precision breeding applications in this commercially important cultivar.
