New approaches to the study of antheridiogen sensitivity in Ceratopteris richardii

The pheromone antheridiogen promotes differentiation of males in some fern gametophyte. Percentages of males in multispore cultures reflect antheridiogen sensitivity in homozygous strains of Ceratopteris richardii. Two homozygous strains (176D and H) with different sensitivities to antheridiogen were genetically characterized by Scott and Hickok (1987). They demonstrated that the difference in sensitivity was associated with either one or two linked genes. The present study was based on this work and had three goals: 1) improve the antheridiogen source by concentrating and partially purifying a crude aqueous filtrate (CAP); 2) develop better techniques for assessing antheridiogen sensitivity; and 3) distinguish between the two models proposed earlier or suggest others.

Extraction techniques used for gibberellic acid were used to partially purify CAE. Concentrated antheridiogen purified by extraction (CAPE) was from 112 to 170 times as active as CAE when autoclaved. If not autoclaved, CAPE exhibited reduced activity. Assays of CAPE effects on gametophytic growth indicated that it did not affect germination timing or reduce overall growth. Spectrophotometric analysis indicated that the purification procedure reduced the heterogeneity of this substance. Strains 176D and H were characterized on CAPE supplemented media with higher antheridiogen concentrations than previously possible.

A new technique was described for assessing antheridiogen sensitivities of individual gametophytes. This involved growing gametophytes in darkness on CAPE supplemented medium and counting the antheridia they produced (dark antheridiogen response). Strains with different antheridiogen sensitivities exhibited characteristic dark antheridiogen responses. This technique when applied to hybridderived spore collections, allowed determination of segregation patterns.

F2s from a cross between 176D and H were characterized on GAPE supplemented media in the light and in darkness. Results indicated that at least four response classes were present. Dark antheridiogen responses determined for several hybrids indicated that 176D and H differ in their antheridiogen response by two linked genes separated by as much as 36 map units.

Characterization and genetic analysis of a mutant which exhibited a high degree of dark germination (>95%) was also described. Darkgermination is associated with a single nuclear gene which functions at the gametophyte level.