Confocal laser microscopic observation of the effects of culture environment on somatic embryo formation

利用共聚焦激光显微镜对荧光-3和Fra Red荧光进行比值分析，测定了胡萝卜体细胞胚发生过程中Ca^<2+>的浓度。同样，由于Ca^<2+> -比值随pH值的变化，pH值也使用carboxy-SNARF-1作为荧光探针进行比例测量。改变培养基pH值对胚前细胞团（PEMs）和体细胞胚的pH值没有影响，但对Ca^<2+>-比值有影响。温度对Ca^<2+>-比值也有影响。除第1天pH为6.0（第0天为转移第3天）外，将PEMs转移到胚胎分化培养基中至第9天pH稳定（约6.8）。[Ca^<2+>]从0.1 μmolL^<-1>（第0天）逐渐下降到0.04 μmolL^<-1>（第5-7天）以下，并在第9天急剧上升到0.4 μmolL^<-1>，第9天首次观察到球形胚。对于由于反复传代培养而失去胚胎发生潜力的细胞团，pH和[Ca^<2+>]始终低于胚胎发生细胞团的值。未观察到[Ca^<2+>]的迅速增加。

Concentrations of Ca^<2+> during carrot somatic embryogenesis was measured based on the ratiometric analysis of fluo-3 and Fra Red fluorescence using a confocal laser microscope. Similarly, pH was also ratiometrically measured using carboxy-SNARF-1 as a fluorescence probe, because Ca^<2+> -ratio varied with pH. The following points were elucidated.1. Changing the culture medium pH did not alter pH in pro-embryogenic cell masses (PEMs) nor in somatic embryos, but influenced the Ca^<2+>-ratio. Also the temperature affected the Ca^<2+>-ratio.2. Stable pH (about 6.8) was observed after transferring PEMs to embryo differentiation medium up to day 9 except for day 1, on which pH of 6.0 was observed, where day 0 is the transferring day.3. [Ca^<2+>] gradually decreased from 0.1 μmolL^<-1> (day 0) to below 0.04 μmolL^<-1> (day 5-7) and sharply increased to 0.4 μmolL^<-1> on day 9 on which globular embryos were first observed.4. In the case of cell masses which have lost embryogenic potential due to repeated subculturing, pH and [Ca^<2+>] were always below the values observed in the case of embryogenic cell masses. No prompt increase in [Ca^<2+>] was observed.