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In angiosperm plants, the FGU refers to the minimum complement of cells required to accomplish double fertilization in vivo (Dumas et al. 1984; Huang and Russell 1992). Typical FGU consists of an egg, two synergids and a central cell, which fulfills the role of pollen tube attraction, sperm discharge to the receptive cells, transportation of sperm nuclei and double fertilization (Russell 1992;Dumas and Mogensen 1993; Higashiyama et al. 2001). Since the report of embryo sac isolation by Zhou and Yang (1982), the procedures for isolation and culture of embryo sac and female gametophytic cells have been developed over two decades, which has enabled fundamental and applied studies in the area of reproductive biology, such as mechanisms of recognition, adhesion and fusion of gametes, and cellular and molecular changes of gametes in double fertilization process (Dumas and Russell 1992; Faure 2001; Raghavan 2003). Furthermore, isolation of individual FGUs could be utilized to perform some applied studies such as breeding of novel plants through in vitro fertilization (Zenkteler 1990; Kranz et al. 1991) and gametosomatic hybridization (Sun et al. 1995).Until now, female gametophytic cells have successfully been isolated in several monocot species such as Zea mays (Kranz et al. 1991; 1998) 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 FGU from 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 10 g l Ϫ1 Cellulase Onozuka R-10, 10 g l Ϫ1 Macerozyme R-10, 0.6 M mannitol, 5 mM 2-morpholinoethane-sulfonic acid and 5 g l Ϫ1 potassium dextran sulfate, pH 5.8 with 50-rpm shaking for 2 h at room temperature. Isolated FGUs were distinguished by their specific size and characteristics. Fluorescent staining with 4Ј, 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 0.6 M mannitol using a micropump-connected microcapillary, about 80% of the isolated FGUs were viable for up to 8 h after the isolation, as determining by fluorescein diacetate staining.

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