Isolation and in vitro fusion of egg and sperm cells in Oryza sativa

Protocols for isolation of gametes of Oryza sativa were developed and initial results on in vitro fusion of sperm and egg cells are reported. The best yield of viable sperm cells was obtained when pollen grains were cultured in a medium containing of 1.3 mM boric acid, 3.6 mM calcium chloride, 0.74 mM potassium phosphate, and 438 mM sucrose. Embryo sacs were isolated using cell wall degrading enzyme treatments for 2-5 h followed by mechanical manipulation. The maximum yield (38.2%) of egg cell was achieved when 2% cellulase and 0.55% pectinase were used in the medium. However, the optimum concentration of cellulase and pectinase was found to be 1% and 0.85%, respectively. Fluorescein diacetate (FDA) stain was used to determine the viable sperm and egg cells. The optimal procedures (fusion conditions) for gametes fusion occurred in a medium containing calcium chloride at a concentration of 7 mM (pH 7.5) and the best result obtained (55.5%) in terms of fused gametes, is reported.