Low-temperature treatment of tobacco ovaries improves the isolation, viability, and embryogenic potential of two-celled proembryos

The influence of low-temperature treatment of ovaries on the isolation and subsequent culture of two-celled proembryo in vitro was explored in tobacco. The efficiency of isolation of two-celled proembryos was significantly improved, by an average of 23.47?% after treatment of ovaries at 4°C for 5?C24?h. Cell viability and polarity of two-celled proembryos, as assessed by organelle distribution, growth pattern, and cell wall component distribution, were all well maintained after cold treatment. Furthermore, in vitro cultivated two-celled proembryos divided normally, with timing paralleling that of two-celled proembryos isolated from fresh ovaries. Unexpectedly, the division frequency of two-celled proembryos was significantly improved by cold treatment. These results demonstrate that low-temperature treatment of tobacco ovaries was beneficial for two-celled proembryo manipulation in vitro, by not only improving isolation efficiency but also by promoting cell division and improving the potential for further cultivation.