Bark sections were excised according to the procedure outlined for healthy tissue, 

 except that cortical tissues were divided into rectangular pieces (100-150 mm.^). One- 

 eighth of each rectangle was infected tissue, seven-eighths of each piece, healthy 

 tissue (f ig . 7) . 



Seventy-five to 98 percent survival and subsequent growth of healthy and blister 

 rust-infected white pine (Harvey 1967; Harvey and Grasham 1969b) and a minimum of 75 

 percent survival and growth of other species (Harvey and Grasham 1969a) were obtained 

 on artificial media by following these techniques. 



DISCUSSION 



The delay in tissue development experienced during the winter months appeared to 

 be related to dormancy. The dormancy requirement apparently had not been satisfied. 

 As a result, the physiological condition of this tissue was less receptive to auxin- 

 induced growth and an adjustment period (not required by active tissue) was necessary. 



The reduced surface oxidation and subsequent increase in growth noted when cultures 

 were started at the beginning of the cool period were apparently related to the effects 

 of temperature on the rate of chemical oxidation. The lower temperature may also have 

 permitted the tissue to neutralize the effects of oxidation. 



The use of large primary explants assures: (1) substantial food reserves neces- 

 sary to maintain the explants until proliferation begins; and (2) reduction of the 

 in jured : uninjured tissue ratio to minimize the effects of damages incurred during 

 tissue preparation. 



The one-eighth infected to seven-eighths healthy tissue ratio used to culture rust- 

 infected tissue allowed for good host development and adequate inoculum potential for 

 rust proliferation. This ratio is important to successful culture of this host-parasite 

 system and produces optimum results. 



The increase of growth experienced when plastic vessel closures were used was ap- 

 parently related to: (1) increased light transmission; (2) the conservation of moisture 

 in the vessel; and (3) an adequate exchange of gases. The requirement for a balanced 

 system of gaseous exchange and moisture conservation was effectively demonstrated when 

 closures permitted either excessive moisture loss (cotton) or no gas exchange (screw 

 caps or corks) . 



Since all other cultural conditions were identical, the increased growth obtained 

 in 125-mli. flasks over that in 25 X 100 mm. tubes was due to the amount of medium 

 and/or gas volume available to the tissues. 



In our experience, failures of primary explants to proliferate into callus tissues 

 were usually related to one or more of the following procedural errors: 



1. --Improper selection of plant materials; 



2. --Delay in excision of tissues after collection of materials; 



3. --Improper length of time for sterilization; 



4 . -- Inadequate removal of a sterilizing agent; 



5 . -- Incomplete removal of the epidermis; 



6. --Damage to cortical tissues from hot or dull excision blades; 



7. --Bruising or tearing of the excised tissues; 



8. --Delay in transferring excised tissues to a culture medium; 



9. --Improper incubation conditions; 



10. --Incorrect selection or concentration of auxin; and 



11 . --Improper pH of the medium. 



4 



