Synchronization based on precise cell cycle progression identification to enhance tetraploid induction in '84 K'...

This research develops a novel method for improving tetraploid induction efficiency in vitro by precisely identifying key cell cycle phases and optimizing synchronization treatments in '84 K' poplar. Cell cycle asynchrony is a critical bottleneck in tetraploid in vitro induction efficiency. 5-Aminouracil (5-AU), a known cell cycle inhibitor, provides a strategy to enhance tetraploid induction efficiency. However, as 5-AU primarily targets S-phase cells and demonstrates cytotoxicity, improving tetraploid induction efficiency requires first accurately identifying the peak window of S-phase cell accumulation in the cultured tissue. Additionally, it is crucial to determine the appropriate concentration range of 5-AU to minimize toxicity to the cultured tissue. In this study, we developed a cell cycle synchronization strategy and evaluated its effect on tetraploid induction efficiency. Flow cytometry and EdU fluorescence labeling were used to track the cell cycle progression of '84 K' poplar (Populus alba × P. glandulosa) leaves during in vitro culture. Our results showed that S-phase activity peaked between days 2 and 3 of differentiation, while the G2/M phase began on day 4. This defined the optimal synchronization window as days 2 to 3. Comet assays were conducted to evaluate DNA damage induced by 5-AU. The results showed that DNA fragmentation increased significantly at concentrations exceeding 1.7 mM. In contrast, at concentrations below 1.3 mM, comet assays indicated relatively low DNA damage, while flow cytometry analysis revealed suboptimal synchronization efficiency. Consequently, 1.5 mM was identified as the optimal concentration for synchronization induction. By applying this synchronization strategy, the tetraploid induction rate of '84 K' poplar was increased to an average of 40.90%. Our results demonstrate that precise synchronization of the cell cycle, with minimal cytotoxicity, can significantly improve tetraploid induction efficiency.