Seedlings from a single lot of Digitalis purpurea L. seeds were germinated in batches over a period of 13 months. A total lipid extract was made which was resolved into esterified and unconjugated plus glycosylated sterol fractions. The amounts of sterol in each fraction and in the total were compared for seedlings germinated at different times of the year. The amount of esterified sterols reached a maximum value from March until June, and a low value from July until January. In Januay, a sharp increase began which lasted until March. Amounts of unconjugated and glycosylated sterols were elevated from March until June, low from July until October, and on the rise from November until March. These data correlate with an annual cycle in seed germination. The phase of maximum sterol content of seedlings is foOowed by a period of null germination.An annual variation in the levels of terpenes and volatile oils in the leaves of several gymnosperm species growing in the field has been reported (1, 10-12). In each case, the lipid levels are higher during the winter months than during the summer. In this paper, we describe the presence of an annual cycle in the sterol content of Digitalis seedlings germinated under controlled laboratory conditions. The cycle correlates with a seasonal fluctuation in the germination of Digitalis seeds, which we have observed for a number of years. The pattern of lipid variation observed in the gymnosperms and in Digitalis suggests an adaptive role which may enable plants to survive periods of low temperature.
EXPERIMENTAL PROCEDURE
MATERIALSSeeds of Digitalis purpurea var. gloxinoides were obtained from Burnett Bros, Inc., New York, N. Y. All reagents were analytical grade. Solvents were distilled prior to use except petroleum ether (boiling range 60 to 90C, Skelly Oil Co., Kansas City, Mo.) and diethyl ether. Cholesterol and stigmaster6l were obtained from Aldrich Chemical Co., and recrystallized from methanol until chromatographically pure. Sterols were separated and quantitated on a Glowall model 320 gas chromatograph equipped with a 183-cm glass column of 3.4-mm diameter, a flame ionization detector, and a Barber-Coleman recorder. The stationary phase was 3% OV-1 coated on Gas 1 This work was done in partial fulfillment of the requirements for the degree of Doctor of Philosophy at Bryn Mawr College by M. K. J.2 Present address: Department of Biology, Beaver College, Glenside, Pa. 19038. Chrom Q, 80 to 100 mesh (Applied Science, State College, Pa.); carrier gas was argon at a pressure of 14 p.s.i. Column temperature was 250 C, flash heater, 250 C, and the detector, 260 C, set at 360 v.
METHODSThree hundred mg of Digitalis seeds (approximately 4000) were grown aseptically in covered Pyrex glass culture dishes, 10 cm in diameter and 8 cm deep. Seeds, stored in the dark at 23 C prior to germination, were sown under artificial white or green light. A layer, 0.5 cm deep, of clean, dry beach sand was deposited in each dish. Dishes were covered and sterilized in a gas oven at 160 C for...