MICROMANIPULATION OF MALE AND FEMALE GAMETES OF NICOTIANA-TABACUM .1.ISOLATION OF GAMETES

The isolation of male and female gametes is a precondition for the mic romanipulation of flowering plant gametes. To reflect their condition at fertilization, isolated gametes need to be physiologically mature a nd vigorous. Sperm cells are isolated from pollen tubes grown on cut s tyles using the ''in vivo/in vitro'' technique. Embryo sacs are isolat ed 2 days after anthesis using brief treatments of minimal concentrati ons of cell-wall-digesting enzymes on ovules of emasculated flowers. E gg cells are then mechanically separated from the embryo sac, allowing unambiguous identification of cells. Two days is usually the mini mum required for the pollen tube to penetrate the ovule and effect fertil ization in vivo.