An in vitro bioassay has been developed to explore the role of the pollen coating in the pollen/stigma interaction in Brassica oleracea. In the assay, coating is removed from pollen grains, supplemented with protein fractions isolated from coatings of different S (self incompatibility) haplotypes, and then—using micromanipulation—interposed between individual pollen grains and the stigmatic surface. Normally, the coating used is of the same haplotype as the pollen in the experiment—thus constituting an `extension' of its own coat—but carrying the supplemented protein fractions. Initial experiments confirmed preliminary data that the pollen coating contained the male determinant of self incompatibility (SI); not only did the addition of `self' coating (i.e. that with the same S-haplotype as the stigma) prevent the success of a compatible cross pollination, but a `cross' coating (i.e. that with a different S-haplotype from the stigma) could induce the germination and growth of self pollen. Protein supplementation experiments demonstrated that the pollen-held determinant is contained within the water soluble component of the pollen coat, while further analysis revealed that the active molecular species possesses an Mr⩿10 kDa. More extensive fractionation by gel filtration and reverse phase HPLC was used to isolate a family of basic, cysteine-rich proteins (PCP-A: Pollen Coat Proteins-class A)—one of which is known to bind to stigmatically-expressed components of the S-locus in Brassica. Introduction of the PCP-A protein fraction into the bioassay confirmed the male determinant of SI as a protein, and probably a member of the PCP-A protein family.
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