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

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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.