Improved mating efficiency inAcetabularia acetabulum: recovery of 48–100% of the expected zygotes

Summary

We have improved zygote recovery 11–1,000 fold by optimizing the physiology of gamete release and mating inAcetabularia acetabulum. Gamete release was affected by agar purity, concentration, and volume/gametangial pair. Cold pre-treatment of gametangia (14–30 d at 10°C in the dark) synchronized subsequent gamete release at 21°C in the light. Cold pre-treatment was nearly twice as effective in synchronizing subsequent gamete release when intact, gametangia-bearing caps rather than isolated gametangia were pretreated. Synchronizing gamete release doubled mating efficiency. In a wild-type laboratory strain ofA. acetabulum, there were 1,561±207 gametes/gametangium which had half-lives of 14.5 d in 0.1% seawater-agar. We recovered 48–93% of the expected numbers of zygotes from a mass mating of 8 to 1,226 gametangia and 11–128% of the expected numbers of zygotes from mating single gametangial pairs: the large range in the calculated mating efficiency may be attributable to the variation in the numbers of gametes made per gametangium. Zygote recovery from single gametangial pairs was highly dependent on the volume of mating matrix. In addition, most zygotes recovered were unattached to any other zygotes in the subsequent generation (> 95% single cells from matings of 1–500 gametangial pairs). Our improvements in mating conditions and zygote recovery (1) have facilitated cell manipulation and culture ofA. acetabulum in the laboratory; and (2) have made controlled crosses for selection and genetic analysis of mutants feasible. These advances have removed a major barrier to genetic analysis of development inAcetabularia.