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Immunofluorescent Localization of Myosin on the Sperm Cells of Plumbago Zeylanica

Sperm cells in flowering plants are non-motile and are passive participants in their movement to the female reproductive cells. It is believed that actomyosin interaction may play a key role for sperm cell transmission in the pollen tube as well as in the embryo sac. However, indirect evidence has shown that the surface of sperm cells lacks amounts of myosin sufficient to support movement. Immunofluorescence microscopy was used in this study to further assess the presence of myosin on the surface of sperm cells of Plumbago Zeylanica. Sperm cells of Plumbago Zeylanica were isolated according to published methods. Isolated sperm cells were blocked 20 min with 1% bovine serum albumin and 2% normal goat serum in phosphate buffered saline (PBS) (pH 7.3, in 15% sucrose), incubated 2 hrs in anti-myosin antibody (M-7648, Sigma Chemical Co., St. Louis, MO) diluted 1:10 with blocking solution, washed three times (5 min) with blocking solution and incubated 1 hr in 1:30 FITC-conjugated anti-rabbit IgG as secondary antibody (EY Labs, Inc., San Mateo, CA) in blocking solution. Samples were rinsed in PBS and mounted in an anti-fading solution with 1:1 PBS:glycerol with 3% n-propyl gallate.