An average of 540 picomoles of total adenosine phosphates was found in the embryo of mature seeds of ponderosa pine (Pinus ponderosa Laws.) and 1140 picomoles in the gametophyte. Adenylate energy charges were 0.44 and 0.26, respectively. After stratification, total adenosine phosphates increased 7-fold and 6-fold in embryo and gametophyte, respectively, and energy charges rose to 0.85 and 0.75. During germination, total adenosine phosphates increased to a 20-fold peak on the 9th day in gametophytic tissue, parallel with the peak of reserve regradation and organellar synthesis, and then decreased. In embryo and seedling, total adenosine phosphates elevated 80-fold with two distinct oscillating increases of AMP and ADP. (Table I). After the seedling length was measured, the seed coat was removed and the gametophyte was dissected. Fresh and dry weights (dried at 100 C for 24 hr) were determined on gametophytes and embryos or seedlings for each replication. Water content was calculated from the difference of fresh weight and dry weight.Determination of Adenosine Phosphates. Four seeds of the designated stage of germination were dissected into gametophyte and embryo or seedling in a moist chamber. The dissection of each seed took less than 10 sec in skilled hands. Each part was quickly extracted by 10 ml of boiling, glass-distilled water for 10 min at 100 C. The extract was cooled in an ice bath, and 0.8 ml of the original (early stages) or diluted (later stages) extract was incubated at 30 C for 15 min in each of the following mixtures (8).A. For ATP determination, 0.1 ml of reaction buffer containing 0.5 M N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES), pH 7.5, and 0.5 M magnesium acetate and 0.1 ml of glass-distilled water. B. For ADP and ATP determination, 0.1 ml of the reaction buffer and 0.1 ml of solution containing 20 ,ug of pyruvate kinase (EC 2.7.1.40) (Sigma, crystalline), and 500 nmoles of trisodium phosphoenolpyruvate (Calbiochem).C. For TAP, 0.1 ml of the reaction buffer, and 0.1 ml of solution containing 20 ,ug of pyruvate kinase, 500 nmoles of trisodium phosphoenolpyruvate. and 20 ,.tg of dialyzed (against 1 mM phosphate buffer, pH 7.0) adenylate kinase (EC 2.7.4.3) (Sigma).After incubation, the extracts were either stored at 0 C overnight or assayed immediately by the luciferin-luciferase method using an Aminco Chem-Glow photometer (24

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Douglas‐fir [Pseudotsuga menziesii (Mirb.) Franco] pollen was germinated and grown in mass in a sterile mineral medium supplemented with 0.3 M mannitol as osmotic stabilizer. During the 4‐day period, pollen elongated threefold; free sugar and amino acid contents did not change significantly; soluble protein, insoluble protein, and RNA decreased; starch reduced to 30%; and DNA doubled in quantity. Respiration rate remained high and constant during the first 36 h and then increased, but the respiratory quotient averaged about 1.0 throughout the period. The pool size of adenine nucleotides remained constant, whereas ATP content and energy charge increased rapidly during the first 8 h and remained high for the remainder of the period. 14C‐glucose was rapidly metabolized within 2 h at 25°C to amino acids (33%), organic acids (22%), sugars (20%), CO2 (15%), lipids (5%), and insoluble components (3%). Experimental data indicated that the cultural conditions provided a suitable environment for rapid germination and active metabolism of Douglas‐fir pollen.

Many biological materials have been treated by freeze-drying for safe storage (2, 5, 13), and preserving pollen by this method has been satisfactory with many species (4, 9, 10, 15). Viability of Douglas fir pollen was reduced by freeze-drying, the extent of the reduction depending on conditions immediately following collection of the pollen and on the duration of freeze-drying. There were no apparent differences in ability to set seed, in germinability of the seed, and in growth of seedlings between seeds from wind pollination and artificial pollination with pollen that had not been freeze-dried, or had been freeze-dried for various periods (11). Air-drying or cold storage immediately following collection of the pollen appeared to be less damaging than direct freeze-drying of fresh material in that study. The mechanism of killing by freeze-drying has been explored very little. Permeability of the cellular membrane of freeze-dried pollen was examined by soaking the pollen in glass-distilled water and measuring conductivity of the filtered leachate at various intervals. Close correlation was noted between germinability of pollen and conductance of the leachates. In addition to total electrolytes, phosphorus-containing compounds, reducing sugars, and amino acids were found by thin-layer chromatography in the leachate. The leachate had a maximum absorption at 260 m,u. Conductance, absorbance at 260 m,u, and quantity of Pi, soluble sugars, and free amino acids were measured on leachates from the same stored pollen freeze-dried for various lengths of time and used to determine germination and moisture content.In the quantitative procedure followed, a sample of 250 mg pollen was soaked in 40 ml of glass-distilled water (specific conductance < 2 X 10-6 mho) at room temperature (22°-25°)