Flavonoid Synthesis and Antheridium Initiation in Dryopteris Gametophytes

Abstract

There is now a fairly extensive vascular crytogam flavonoid literature and there are a number of researchers actively engaged in this research field (Swain & Cooper-Driver, 1973). All vascular crytogam flavonoid work has been done on the sporophyte generation, with one exception: Laurent (1966) determined that Blechnum brasiliense gametophytes produced the flavonoid kaempferol, which is one of the flavonoids produced by the B. brasiliense sporophyte. Reasons for the paucity of information on fern gametophyte flavonoids include the easy accessibility of sporophytes and the now disreputed opinion that flavonoids, being associated with lignin synthesis, are exclusive to vascularized plant bodies. This exclusivity has been lost because flavonoids have been isolated and identified in various non-vascular plant groups: certain algal divisions, bryophytes (Swain, 1974), and fern gametophytes (Laurent, 1966). Initially our investigation was undertaken to determine if Dryopteris intermedia A. Gray and D. marginalis A. Gray gametophytes produce flavonoids and, if so, were these the same flavonoids produced by their sporophytic counterparts (Petersen, 1976). Because of the unusual results of this first portion of the research, the inquiry was amplified to include an analysis of flavonoid content along a developmental profile of the gametophytes. Half-strength White's minimum nutrient medium adjusted to pH 6.0 was used to culture the gametophytes. Liquid cultures were prepared by placing 0.25 g of spores in a 4 1 flask and adding 2 1 of nutrient solution. Separate cultures of D. intermedia and D. marginalis were grown at 22?C under 300 ft-c of illumination from cool-white fluorescent lights in a 12/12 hr diurnal cycle. Gametophytes were harvested and assayed for flavonoids at three developmental stages: (1) pre-antheridial initiation (0 antheridia/gametophyte), (2) antheridial initiation (0 or 1 antheridia/gametophyte), and (3) post-antheridial initiation (4-6 antheridia/gametophyte). Ten-gram samples of harvested gametophytes were immediately extracted in methanol and re-extracted repeatedly until a colorless supernatant was obtained. Concentrated extracts were spotted onto Whatman 3MM chromatography paper (42 x 55 cm). Chromatograms were developed in two dimensions employing the two standard solvent systems for the separation of flavonoids: t-butanol, acetic acid, water (3:1:1) for the first dimension and 15% acetic acid, water for the second dimension. Completed chromatograms were inspected under UV light, both in the presence and absence of NH3. Spot color changes under both conditions were noted and R tvalues determined. Spots were excised, eluted in spectral grade methanol, and UV spectral data obtained using standard procedures (Mabry