Micromanipulation of Protoplasts from Female Gametophytic Cells in Common Buckwheat (Fagopyrum esculentum Moench.)

Abstract

The isolation of female gametes is precondition for the satisfactory micromanipulation of gametes in common buck-wheat (Fagopyrum esculentum Moench.). Embryo sacs were isolated at flowering stage after brief treatment with minimal con-centrations of cell-wal-digesting enzymes on ovules. Afterward, egg cells were mechanically separated from embryo sac, allowingunambiguous identification of cells. The optimum isolation solution contains 9% mannitol, 0.75% pectinase, 0.25% pectolase and0.5% celluase at pH 5.8 and 9% osmolality with shaking at 100 rpm. Protoplasts derived from each embryo sac were connectedtogether after isolation or aggregated with egg cell protoplast and two synergid protoplasts were viable even until two hours afterisolation. Egg cells were isolated from embryo sacs by osmotic shock. Right combination of pH and osmolailty were important foroptimal isolation. Notable diferences between somatic and egg cells included the absence of a filiform apparatus and the accu-mulation of dense particles in the plastids, which was most conspicuous in eg cells damaged during isolation. The significantimportance of isolated protoplasts of egg cells was also discussed with respect to genetic manipulation, i.e., target gene conversionor crossing for gene.