Cut sections were washed for 1 hour under rapidly running tapwater to free them 

 of dirt and other foreign matter. In our laboratory, a longer washing period resulted 

 in contamination by bacteria commonly associated with the water supply. Water-saturated 

 stem sections were not satisfactory to tissue culture for this reason. 



Sterilisation. --After being washed, the sections were gently shaken in 200-500 mSL. 

 of a 5.25% sodium hypochloride (NaOGl) solution contained in a 1-liter, screw-top flask. 

 No more than 25 sections were treated at one time in order to minimize the total contam- 

 inant load and to assure proper sterilization. If stem sections were properly chosen, 

 they required only 1 to 3 minutes of surface sterilization. Since the cortex is well- 

 protected by the unbroken epidermis of current-year stems, it was relatively free of 

 contamination and only surface sterilization was necessary. An alternate treatment, the 

 use of a 3% hydrogen-peroxide (H2O2) solution for 24 hours, successfully controlled con- 

 tamination of conifer species (Pinus nigra^ P. ponderosa^ Larix oaoidentalis ) easily 

 damaged by the NaOCl solution (Harvey and Grasham 1969a) . Whenever cortical tissues 

 had low levels of surface contamination, decreasing sterilization periods resulted in 

 better tissue culture development, although for most tissues more time was needed to 

 control contamination. An increase in sterilization delayed callus development. Imme- 

 diately following sterilization with either NaOCl or H2O2, stem sections were rinsed 

 three to five times in cool, sterile distilled water to remove all traces of the 

 sterilant. (Complete removal of the sterilizing agent is necessary for normal callus 

 development .) 



Excision of primary expZants . --Immediately after sterilization, cortical tissues 

 were aseptically excised from the stem sections and transferred to the desired medium. 

 These tissues constituted the primary explants. 



During the excision process, all tools were kept cool (room temperature), extremely 

 sharp, and free of the sterilizing agent. Razor blades and scalpels were changed fre- 

 quently to prevent dull or damaged tools from tearing or bruising the tissue. Bruising 

 or tearing of the cortical tissues during excision caused discoloration and abnormal 

 development of the callus tissue. Similar effects were observed when tissues came in 

 contact with hot instruments or with a sterilizing agent. 



Each stem section was separately removed from the flask in a sterile room and 

 placed on a sterile surface. Each section was held gently but firmly by sterile forceps. 

 A sterile blade was then used to make a longitudinal slit through the bark and cambium 

 (fig. 4). The Bard Parker surgical blade No. 11 was satisfactory. Forceps were then 

 used to rotate the section while the cuticle and epidermis were surgically removed in 

 thin, longitudinal strips (fig. 5). (Several fungi are associated with these tissues; 

 so complete removal of these layers is essential .) Extreme care was taken not to bruise 

 cortical tissues during this process. Cortical tissues were then undercut in the cam- 

 bial zone (fig. 6) and carefully lifted from the woody cylinder (fig. 7). The wood was 

 discarded. The damaged ends of the cortical tissues were trimmed, and the remaining 

 tissues cut into 100-150-mm.^ rectangular pieces. These pieces then were placed, cam- 

 bium side down, on culture media. Optimum tissue culture growth and callus prolifera- 

 tion were obtained when large (100-150 mm.^) primary explants were used. All working 

 surfaces and tools were resterilized with alcohol, flame-dried, and air-cooled before 

 the next section was prepared. 



Incubation vessels . --Standard flare-mouth culture tubes (25 X 100 mm.) covered 

 with a single layer of plastic food wrap were used successfully as incubation vessels. 

 The tubes contained 15 m£. of medium and proved to be satisfactory for tests of 90 days' 

 duration. Similarly covered, 125-m2.., wide-mouthed Erlenmeyer flasks, containing 50 m2.. 

 of medium, produced superior growth when cultures were maintained more than 90 days. 



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