Isolation of individual egg cells and zygotes in Alstroemeria followed by manual selection with a microcapillary-connected micropump.

AIMS: To develop a procedure for isolating living egg cells and zygotes from Alstroemeria ovules. SCOPE: An attempt was made to isolate egg cells and zygotes from the ovules of Alstroemeria aurea. The ovules were histologically observed using a clearing procedure which revealed the localization and sizes of the embryo sacs and egg apparatus within the ovules. For the isolation of egg cells, ovules were cut into sections with a surgical blade and treated with an enzyme solution. Subsequently, these ovule sections were dissected using a glass needle under an inverted microscope. Egg cells successfully isolated by this procedure were collected using microcapillaries connected to a micropump. For zygote isolation, ovules were excised from ovaries 24 h after self-pollination. By treating excised ovules with an enzyme solution and subsequently dissecting them using a glass needle, zygotes were successfully isolated from the ovules and collected with a microcapillary. The isolated zygotes were associated with pollen tubes and one of the synergids. Egg cells and zygotes were viable for up to 2 h following isolation, as determined by fluorescein diacetate staining.
CONCLUSIONS: The procedures for isolating egg cells and zygotes in Alstroemeria were established, and each egg cell and zygote was captured with a microcapillary.