Isolation and collection of two populations of viable sperm cells from the pollen of Plumbago zeylanica.

A protocol is described for individually collecting two populations of sperm cells, Svn and Sua, from pollen of Plumbago zeylanica. Pollen grains were burst in 10 mM MOPS buffer containing 0.8 M mannitol (pH 4.6). Paired sperm cells released from pollen were separated using a microinjector. Svn and Sua were then collected individually with a microinjector, based upon known size differences. Collected sperm cells were washed with isolation medium and transferred to liquid nitrogen until use. Fluorochromatic reaction (FCR) test of isolated sperm cells showed a positive reaction, indicating that the isolated sperm cells are viable; most of the sperm cells retain viability for at least 2 h.