IN-VITRO POLLEN-TUBE GROWTH AND PENETRATION OF FEMALE GAMETOPHYTE IN DOUGLAS-FIR (PSEUDOTSUGA-MENZIESII)

Pollen tube and female gametophyte interactions in Douglas fir (Pseudo tsuga menziesii) were examined in vitro. Formation of pollen tubes in Douglas fir occurred on a modified Murashige and Skoog medium in which concentrations of H3BO3 and Ca(NO3)(2) were altered and supplemented with sucrose and polyethylene glycol. Addition of 100 mu g/ml H3BO3 an d 300 mu g/ml Ca(NO3)(2) resulted in optimum pollen viability. Lack of H3BO3 inhibited pollen tube formation. Addition of H3BO3 and Ca(NO3)( 2) significantly increased pollen tube formation within one week in cu lture. Using a medium supplemented with mannitol, viability of Douglas fir pollen can be sustained for 7 weeks in culture, about the same le ngth of time as in vivo. However, pollen tubes are not formed. This su ggests that the factors responsible for tube formation reside in the e xternal environment of the pollen. Culture of female gametophytes to e xamine egg viability and longevity had not been done previously. We fo und that egg viability in culture is short-lived, and therefore the wi ndow to study and manipulate events of fertilization in Douglas fir is very limited. In spite of this, about 7% of the female gametophytes t hat were cocultured became penetrated by pollen tubes. In vitro archeg onial penetration has been repeatedly achieved, but pollen tubes also penetrated other parts of the female gametophytes. Pollen tube also pe netrated non-viable eggs. Most female gametophytes were not penetrated non-viable cause of pollen tube branching and swelling, failure of tu bes to orient towards the female gametophytes, or premature pollen tub e death due to plasmolysis. This report outlines the first attempt tow ards in vitro fertilization in conifers.