INTRODUCTION 



The tissue culture technique has long been used to study the physiology of specific 

 plants, plant organs, and tissues. By employing this technique, one can control the 

 environment, the substrate, and (if he uses clonal stock) the genetic composition of 

 the tissues under observation. Thus, the effects of variables on such studies can be 

 minimized or eliminated. 



It is recognized that members of the order Coniferales are difficult to propagate 

 vegetatively in vitro (Loewenberg and Skoog 1952; Reinert 1962). Researchers have re- 

 ported excessive tissue damage and/or secondary contamination during the primary explant- 

 ing stage (Gautheret 1959; Koenigs 1968). In the authors' opinion, failures to culture 

 conifer tissues have been due to improper preparative techniques and tissue-selection 

 criteria. Our success with several conifer species (Harvey 1967; Harvey and Grasham 

 1969a) and with rust-infected western white pine, Pinus monticota Dougl., (Harvey and 

 Grasham 1969b) prompted us to prepare this paper detailing improved techniques. 



TECHNIQUES 



To be successful, our techniques require timing as well as treatment; so we have 

 outlined them in order, from sample selection through explant incubation. 



Preparation of Healthy Conifer Tissues 



Tissue-selection criteria. --Optimum tissue culture proliferation was obtained by 

 using primary explants from current-year stems of either vigorous nursery stock or field- 

 grown trees. Satisfactory results were also obtained by using older material, but callus 

 development and growth were not as rapid. Callus development was delayed when we cul- 

 tured material collected in Idaho during the months of December, January, and February. 



Contamination was kept minimal by using stem sections that had a smooth bark, free 

 of insect or mechanical damage (figs. 1 and 2). Except for the fungi and bacteria in- 

 digenous to the cortex, subepidermal bark tissues were relatively free of contamination. 



Cultures were prepared the day that material was collected. Although overnight 

 refrigeration of material before preparation was acceptable, further delay allowed tis- 

 sue oxidation and contamination buildup, both of which are severely detrimental to in 

 vitro tissue growth. 



Pre-sterilization preparation. --Needles were clipped flush with the bark surface 

 before the stems were sterilized. Care was taken to avoid damage to bark tissues dur- 

 ing this operation. Tears permitted the sterilizing fluids used during subsequent 

 operations to penetrate and injure cortical tissues. Tissue damaged by excessive 

 penetration of sterilizing fluids proliferated poorly in vitro. 



Stems from which needles had been removed were sectioned into 2-3-cm. lengths by 

 means of a high-speed, fine-bladed jigsaw (fig. 3). The fraying action of the recipro- 

 cating blade tended to seal the open ends, which prevented excessive penetration by 

 sterilizing fluids. End pieces were extensively damaged at the time of collection and 

 were always discarded. 



