The plastic film closures replaced Dispo foam plugs that were used during prepara- 

 tion of the media and as closures until all primary explants had been placed on a cul- 

 ture medium. 



Closures were prepared by pressing large sheets of plastic food wrap on an alcohol- 

 dampened surface for sterilization. Sheets then were cut in place with a razor blade 

 into 6-cm. squares and placed, sterile side down, over the mouth of the vessel. A 

 tight-fitting rubber band secured the closure. 



Plastic film closures were superior to other types of closures tested. Tissue 

 cultures grown in tubes that had cork or screw-cap tops showed erratic growth. Bacter- 

 iological stoppers or cotton plugs permitted media to dry too rapidly for our needs. 



Incubation environment. --Explants were incubated at 20° C. ±2° with light for 16 

 hours and then at 5° C. ±2° without light for 8 hours (Harvey and Grasham 1969b). Cool, 

 white fluorescent light at an intensity of 400 ft.-c. was satisfactory. Tissue cultures 

 grown in darkness or semidarkness lacked vigor compared to those grown under light. 

 Optimum results were obtained by beginning incubation of the explants at the start of 

 the cool, dark period of the incubation cycle. 



Mediian. --Good proliferation was obtained with many conifer species (Harvey 1967; 

 Harvey and Grasham 1969a) by using a simple medium consisting of the essential auxins, 

 indoleacetic acid (lAA) , napthaleneacetic acid (NAA) or 2,4-dichlorophenoxyacetic acid 

 (2,4-D), at concentrations between .01 and 5 mg./i. 



Addition to the medium of certain amino acids, vitamins, and other growth factors 

 further enhanced growth and development of many species (Harvey and Grasham 1969a) . 

 Tissue cultures benefited most when such compounds were used in microgram quantities 

 (10, 100, 1,000 \sg./l.) . 



All heat -sensitive organic compounds were sterilized by filtration and added to the 

 autoclaved, basal salt medium (Harvey and Grasham 1969a) after it had cooled to 50° C. 



The pH of the medium played an important role in the initial growth of the cambium 

 layer. Our work indicated that a pH range of 4.0 to 6.0 was suitable for the initial 

 growth of most cambium explants (Harvey and Grasham 1969a). 



Preparation of Blister Rust-Infected 

 Western White Pine Tissues 



Save for minor modifications, these same techniques were used for the in vitro 

 culture of rust-infected western white pine (Harvey and Grasham 1969b). The proper 

 selection of stem sections is critical to the culture of infected material. Material 

 chosen must be 2 to 3 years older than that used in healthy tissue studies to assure 

 rust infection and ramification. Nonfruiting rust cankers from 2- to 3-year-old growth 

 segments of 6- to 10-year-old seedlings have been the best source of infected material. 



After the needles were removed in the laboratory, the stems were sectioned as pre- 

 viously described. Only the outer edge of the infection was used. Stem sections were 

 cut so that they were one-eighth infected and seven-eighths healthy tissue (fig. 3). 



, A sterilization period of 5-10 minutes using 5.25% NaOCl solution was necessary to 

 adequately control contamination. Again, all traces of the sterilant were removed by 

 rinsing the sections three to five times in cool, sterile distilled water. 



3 



