THE MALE DETERMINANT OF SELF-INCOMPATIBILITY IN BRASSICA-OLERACEA IS LOCATED IN THE POLLEN COATING

An in vitro bioassay has been developed to explore the role of the pol len coating in the pollen/stigma interaction in Brassica oleracea. In the assay, coating is removed from pollen grains, supplemented with pr otein fractions isolated from coatings of different S (self incompatib ility) haplotypes, and then-using micromanipulation-interposed between individual pollen grains and the stigmatic surface. Normally, the coa ting used is of the same haplotype as the pollen in the experiment-thu s constituting an 'extension' of its own coat-but carrying the supplem ented protein fractions. Initial experiments confirmed preliminary dat a that the pollen coating contained the male determinant of self incom patibility (SI); not only did the addition of 'self' coating (i.e. tha t with the same S-haplotype as the stigma) prevent the success of a co mpatible cross pollination, but a 'cross' coating (i.e. that with a di fferent S-haplotype from the stigma) could induce the germination and growth of self pollen. Protein supplementation experiments demonstrate d that the pollen-held determinant is contained within the water solub le component of the pollen coat, while further analysis revealed that the active molecular species possesses an M-r less than or equal to 10 kDa. More extensive fractionation by gel filtration and reverse phase HPLC was used to isolate a family of basic, cysteine-rich proteins (P CP-A: Pollen Coat Proteins-class A)-one of which is known to bind to s tigmatically-expressed components of the S-locus in Brassica. Introduc tion of the PCP-A protein fraction into the bioassay confirmed the mal e determinant of SI as a protein, and probably a member of the PCP-A p rotein family.