Role Of Calcium During Double Fertilization In Torenia Fournieri

Functions of Calcium during fertilization of angiosperm is one of research focuses for botanists. Prior studies of calcium on sexual reproduction of angiosperm mainly covered these about pollen germination and tube growth processes; there are, however, few works on pistil tissue. So far, achieved studies on calcium in embryo sac of angiosperm mostly located in memebrane-binding calcium and total calcium but little in free cytoplasmic calcium ([Ca2+]cyt ). Obstacles to progress are : (1) the double fertilization of most angiosperms occurs internally, deep within ovary tissues, as a result it is very difficult to investigate the truth. (2) There are few ideal techniques for mearsuring [Ca2+]cyt. In conclusion, both limitation delay studies of [Ca2+]cyt in embryo sac during fertilization.The thesis work is to investigate the connection of [Ca2+]cyt and fertilization by avoiding drawbacks noted above, say, employing an ideal experimental plant, Torenia fournieri , with a naked embryo sac that protrudes from the micropyle. Methods to be applied include extracellular Ca2+ influx treatment and intracellular Ca2+ measure by transgenical expression of Ca2+ fluorescence indicator during fertilization. The former is first to obtain protoplasts of female germination unit in different development stages, then Ca2+ lonophore treatment; the latter is to express transgenically the Ca2+-sensing cameleon protein in Torenia fournieri, which allows long term measuring stimulus-induced cytoplasmic Ca2+ changes.First we employed C.W.M2R, a fluorescent fabric brightener, to investigate changes of wall of female germ unit before and after fertilization. Before anthesis wall of egg apparatus is complete; most part of egg wall at the chalazal end disappears at the day of anthesis; after frtilization (20h after pollinating) egg wall becomes intact. The results suggest that rebuilding of wall is one of the early cytological events during fertilization. Based on these observations, we isolated protoplasts from female germ unit and performed extracellular Ca2+ influx experiment in order to check whether the cytological event similar to what happens during fertilization will be induced by extracellular Ca2+ influx. The results of extracellular Ca2+ treatment revealed that influx of extracellular Ca2+ induced some cellular event and protoplasts of female germ unit in different development stages respond to the treatment differently. Ca2+ ionophore treatment induced occurrence of new cellulosic wall around protoplasts of unfertilized egg cells from mature embryo sac , which just does occur during fertilization. New wall in protoplasts from fertilized egg cells or from egg cells before anthesis, however, weren't detected, as well as protoplasts of central cell. These results indicated that the increase of cytoplasmic Ca2+ from extracellular Ca2+ influx contributes to the rapid establishment of cellulosic cell wall. Moreover, these results also suggest that only mature unfertilized egg cells, which are at a "special" state, are able to respond to Ca2+ signal to establish new wall rapidly.As for construction of transgenic Torenia fournieri, some foundermantal works have been done including plasmid amplification, Agrobactarium transformation, leaf disk transformation- regeneration, etc.. The work for screening transgenic plant which expresses YC2.1 is in progress.