预培养对尾巨桉DH32-29遗传转化效率的影响OA

[Objective]To obtain a stable and efficient genetic transformation rate,generate a large number of transgenic lines,and create improved varieties,the effects of different preculture treatment durations on genetic transformation of Eucalyptus urophylla×E.grandis DH32-29 were studied.[Method]This study aimed to fine-tune different preculture durations to determine the optimal timing for stable genetic transformation of E.urophylla×E.grandis DH32-29,5-ethynyl-2'-deoxyuridine(EdU)staining and flow cytometry were used to quantify the proportion of cells in the DNA synthesis(S)phase of the cell cycle,and RT-qPCR was used to m easure the expression levels of S phase-related genes EgrCDKD3,EgrCYCD3;3,EgrCYCD1;1,and EgrCYCH1;1.[Result]The duration of preculture of leaf explants significantly affected both the transient and stable transformation efficiency of E.urophylla×E.grandis DH32-29.After 3 days of preculture,the proportion of explant cells in the S phase was the highest,and the expression levels of key S-phase genes EgrCDKD3,EgrCYCD3;3,EgrCYCD1;1,and EgrCYCH1;1 peaked,indicating that explants precultured for 3 days are optimal for Agrobacterium tumefaciens infection.Further stable transformation assays demonstrated that a 3-day preculture substantially improved the efficiency of Agrobacterium-mediated genetic transformation in eucalyptus,increasing the transformation efficiency from 0.77%to 4.67%.[Conclusion]Preculture is crucial for the efficiency of Agrobacterium-mediated genetic transformation in eucalyptus.A 3-day preculture period can effectively enhance the genetic transformation efficiency of eucalyptus.