Towards in Vitro Fertilization in Conifers: Gametophyte Culture and Ineraction, 1997 Biological Sciences Symposium Proceedings

Significant advances have been made towards in vitro fertilization in conifers. Methods for culturing pollen of Douglas-fir were developed. Dissected megagametophytes of known developmental stages were cultured to establish conditions in which they could survive. To introduce the pollen to the megagametophytes, three different approaches were tried. The first involved picking pollen from the ovule and introducing it in vitro to the megagametophyte. This was far less successful than culturing pollen on their own on elongation medium, and then transferring them to a medium which favoured tube growth and upon which megagametophytes with mature eggs had been placed. Most successful was a one-step medium suitable for pollen germination and megagametophyte growth. Penetration of megagametophytes occurred both within species and between species. A series of experiments to establish embryo rescue criteria was carried out. Embryos can be rescued from two weeks after fertilization and onwards, which corresponds to all stages other than proembryo and early embryo.