Cell cycle synchronization in Panax ginseng roots for cytogenomics research

Although molecular cytogenomic data have contributed to elucidating the organization of the Panax ginseng genome, progress in cytogenomics research has been slow because of compounding factors like seasonal availability of seeds as a source of root metaphase chromosomes, low seed yield and germination rate, slow growth, and low mitotic index of the root meristems. To expedite the chromosome analysis, we optimized a system to synchronize the P. ginseng root meristem cell cycle and accumulate metaphase chromosomes from either seedling roots or hairy roots. We analyzed the effects of different concentrations of hydroxyurea on cell cycle synchronization as well as the effects of amiprophos-methyl, oryzalin, and nitrous oxide on metaphase chromosome accumulation. We also analyzed the effect of incubation temperature on the rate of mitosis. We found that the rate of mitosis peaked, with ~ 44% mitotic cells, 13 h after their release from an 18-h treatment with 1.25 mM hydroxyurea at 25 °C. Microtubule inhibition with nitrous oxide starting 2 h before the peak of mitosis produced consistently abundant and well-separated metaphase chromosomes compared with those produced when other microtubule inhibitors were used. We successfully established a cell synchronization and metaphase chromosome accumulation system in P. ginseng. This system will help to expedite the ongoing cytogenomic-aided P. ginseng genome assembly validation and also aid in the isolation of P. ginseng individual chromosomes for future cytogenomics research.