133 Development of desiccation tolerance and long-term preservation after desiccation of suspension-cultured cells of Citrus reticulata

Plant cells are able to acquire tolerance to various stresses such as freezing and desiccation when cultured under suitable controlled conditions.We previously showed the development of a high level of desiccation tolerance ( 2 O/gDW) in suspension-cultured cells of the liverwort Marchantia polymorpha after being cultured in media with high concentrations of sucrose in a study carried out to elucidate the mechanisms of desiccation tolerance and vitrification at the cellular level (Planta 231, 965(2010)).It has been shown that these cells could be preserved at 5 °C for more than 5 years after being desiccated at ambient temperatures.In the present study, we investigated the development of desiccation tolerance and long-term preservation of desiccated cells in suspension-cultured cells of Citrus reticulata .Suspension-cultured cells of C. reticulata “Ohta Ponkan” were precultured in sucrose-enriched (0.25–1.0 M sucrose) media for a total of 5 days and then desiccated on silica gel for 24 h at room temperature.After desiccation for 24 h, water contents of control and precultured cells were 0.03 and 0.06 gH 2 O/gDW, respectively.After desiccation, the cells in a small cup were placed on 0.8% agar medium in a closed container and slowly rehydrated for 24 h and then directly placed on a solidified medium containing 1.0 M sucrose for 24 h before being regrown on the usual medium.Survival rate of cells after desiccation was determined by the TTC reduction test (Steponkus and Lanphear, 1967).The survival rate after desiccation for 24 h of control cells was less than 5%, whereas that of precultured cells was about 80%.Culture of cells at high temperatures (35–45 °C) during preculture for 1 h just before desiccation improved the survival and regrowth rates of cells after desiccation.The effect of high temperature culture was only observed in media with high concentrations of sucrose.Leakage from desiccated cells of cell components that absorbed UV light at 280 nm was observed after rehydration.This leakage of cell components from the cells after desiccation was clearly decreased in cells subjected to high temperature culture.Culture of cells in media with 1.0 M sucrose and high concentrations of calcium (5–20 mM) for 3 h just before desiccation also improved cell survival after desiccation.These effects of high temperature culture and cell culture in media with high concentrations of calcium were more clearly observed in cells in an active growing stage.Culture cells of C. reticulata precultured and then desiccated for 24 h were preserved at 23, 5, and −33 °C for 6 months.The survival rates of cells preserved at −33 and 5 °C were about 100 % and 70 %, respectively, whereas the survival rate of cells preserved at 23 °C was only 25%. Source of funding: This work was supported by Grand-in-Aid for Scientific Research (23658086) from JSPS. Conflict of interest: None declared. sugawara@mail.saitama-u.ac.jp