Comparative Detection of Calcium Fluctuations in Single Female Sex Cells of Tobacco to Distinguish Calcium Signals Triggered by in vitro Fertilization

Double fertilization is a key process of sexual reproduction in higher plants. The role of calcium in the activation of female sex cells through fertilization has recently received a great deal of attention. The establishment of a Ca2+-imaging technique for living, single, female sex cells is a difficult but necessary prerequisite for evaluating the role of Ca2+ in the transduction of external stimuli, including the fusion with the sperm cell, to internal cellular processes. The present study describes the use of Fluo-3 for reporting the Ca2+ signal in isolated, single, female sex cells, egg cells and central cells, of tobacco plants. A suitable loading protocol was optimized by loading the cells at pH 5.6 with 2 mu M Fluo-3 for 30 min at 30 degrees C. Under these conditions, several key factors related to in vitro fertilization were also investigated in order to test their possible effects on the [Ca2+](cyt) of the female sex cells. The results indicated that the bovine serum albumin-fusion system was superior to the polyethlene glycol-fusion system for detecting calcium fluctuations in female sex cells during fertilization. The central cell was fertilized with the sperm cell in bovine serum albumin; however, no evident calcium dynamic was detected, implying that a transient calcium rise might be a specific signal for egg cell fertilization.