EXPERIMENTAL STUDIES AND RESULTS 



Seed Source and Preparation 



The seeds used for experiments during the summer of 1974 were obtained from the 

 U.S. Department of Agriculture Forest Service Nursery at Coeur d'Alene, Idaho. These 

 seeds had been collected from the Seeley Lake Ranger District at an elevation of 

 4,000 feet ± 500 feet. 



During the fall of 1974, approximately 1,000 pine cones were clipped from large 

 ponderosa pine trees on the Blackf oot-Clearwater Game Range, which is adjacent to the 

 Seeley Lake Ranger District, and the location of our field study site where field 

 germination and growth experiments were conducted. The field results will be reported 

 in a separate paper. Approximately 10 pounds of clean seeds were collected. All ex- 

 periments conducted from the fall of 1974 through 1975 used the seeds from this source. 



The seeds in all experiments were soaked in warm water for 24 hours and then placed 

 in 3 percent Chlorox solution for 15 minutes to kill seedcoat pathogens. After soaking 

 in Chlorox, the seeds were stratified at 1° to 5°C for a minimum of 2 weeks. Just 

 prior to use, the stratified seeds were again soaked for 15 minutes in the 3 percent 

 Chlorox solution. 



Tests for Volatile Inhibitors 



Matevials and Methods 



To test for the presence of volatile toxins in pine tissues, green needles, 

 surface litter (dried pine needles) , decomposing duff (decaying pine needles) , roots 

 (1 to 4 cm in diameter), and bark, were collected at the field study site, placed in 

 plastic bags and returned to the laboratory. 



The technique used to test for toxic volatile compounds was similar to that des- 

 cribed by Muller, and others (1964). Sterilized cellulose sponges were brought to 

 maximum water holding capacity by soaking them in distilled water and allowing them 

 to drain. The wet sponges were placed in sterile 100 x 80 mm petri dishes, and each 

 sponge was covered with 7,0 cm filter paper soaked with distilled water. Fifteen pine 

 seeds were then placed on top of the filter paper. 



Two grams of each fresh plant material, described above, were broken up by hand 

 and placed into separate 5 x 2 cm plastic vials. One plastic vial containing plant 

 material (treatment) or no plant material (control) was placed into each petri dish 

 adjacent to the sponge seedbed, permitting only gaseous contact between plant material 

 and seeds. The petri dish was covered and placed randomly into a dark conditioning 

 chamber set at 25°C ± 1°. Five replications were set up for each of the five plant 

 materials and the control. 



Germination was recorded daily during which time the seeds were exposed to 

 approximately 1 hour of light. After germination was completed, the radicle lengths 

 were measured from root tip to seed coat or from root tip to the start of the stem in 



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