Micromanipulation of male and female gametes ofNicotiana tabacum: I. Isolation of gametes.

The isolation of male and female gametes is a precondition for the micromanipulation of flowering plant gametes. To reflect their condition at fertilization, isolated gametes need to be physiologically mature and vigorous. Sperm cells are isolated from pollen tubes grown on cut styles using the "in vivo/in vitro" technique. Embryo sacs are isolated 2 days after anthesis using brief treatments of minimal concentrations of cell-wall-digesting enzymes on ovules of emasculated flowers. Egg cells are then mechanically separated from the embryo sac, allowing unambiguous identification of cells. Two days is usually the minimum required for the pollen tube to penetrate the ovule and effect fertilization in vivo.