Direct isolation of female germ units from ovules of Petunia hybrida by enzymatic treatment without releasing somatic protoplasts from ovular tissue

Abstract

Establishment of the efficient method for isolating the female germ unit (FGU; egg, synergid and central cell) is useful for the studies on the characterization of each FGU as well as in vitro fertilization and gametosomatic hybridization. In this study, an easy one-step enzymatic procedure was successfully developed to isolate FGUfrom ovules of Petunia hybrida without releasing the somatic protoplasts from ovular tissues, which could not be achieved in the previous studies. Each FGU was separately liberated after treating the ovules, which were collected from ovaries of flowers one day after anthesis, with an appropriate enzyme solution comprised of 10gl -1 Cellulase Onozuka R-10, 10gl -1 Macerozyme R-10, 0.6M mannitol, 5mM 2-morpholinoethane-sulfonic acid and 5gl -1 potassium dextran sulfate, pH 5.8 with 50-rpm shaking for 2h at room temperature. Isolated FGUs were distinguished by their specific size and characteristics. Fluorescent staining with 4, 6-diamidino-2-phenylindole could identify the polar nuclei of central cell and the nuclear polarity of egg apparatus cells. After transfer into washing solution supplemented with 0.6M mannitol using a micropump-connected microcapillary, about 80% of the isolated FGUs were viable for up to 8h after the isolation, as determining by fluorescein diacetate staining. © 2009 The Japanese Society for Plant Cell and Molecular Biology.