First part: 1. A deep freezing technique has been developed inDrosophila melanogaster which makes it possible to freeze (1) imaginal discs, (2) blastemas of imaginal discs which have been culturedin vivo and (3) embryonic neoplasms. 2. The suitability of differentsalt solutions for our freezing medium has been tested. It is clear that salt solutions containing organic additives are more suitable than are pure ones. It remains to be determined if, in addition to glycerol, the added sugars also act as freezing protectives. 3. Glycerol as a freezing protective (i.e., cryoprotective) is much better than Dimethylsulfoxide (DMSO) since it was shown not to be toxic to insect tissue. 4. In either the presence or the absence ofprotein in the freezing medium the imaginal discs survive the freezing process. However, without protein the tissue becomes sticky which makes successful transplantation virtually impossible. When the concentration of protein is high (6 mg/ml) in a 10% (v/v) glycerol-freezing medium, the protecting effect is considerably reduced. In contrast, a 10 % (v/v) DMSO-freezing medium requires such a high concentration of protein in order to reduce the toxic effects of the DMSO itself. As protein components, both Fetal Calf Serum (FCS) and Bovine Serum Albumin (BSA) are possible. However, since the concentration of protein in FCS is high and, in addition, the correct dose of protein cannot be measured out, the purified protein BSA is more suitable. 5. The optimalfreezing-velocity for our tissue is 1° C/minute. Thethawing-velocity is 100° C/ minute. 6. The freezing process has no detectable effect on determination and differentiation, nor ontransdetermination.
Second part: 7. The suitability of the technique for freezing imaginal discs has also been tested withlarval ovaries ofDrosophila melanogaster. 8. The normal practice of dipping imaginal discs directly into the freezing medium cannot be applied to larval ovaries. Specifically, under these conditions the membrane of the larval ovary is damaged. Due to this osmotic shock the processes of freezing and thawing lead to the loss of cells and eventual death of the larval ovary. 9. Osmotic shock is not observed if the larval ovaries are stepwise transferred to progressively higher concentrations of cryoprotective in the freezing medium. 10. As a function of the genotype, between 66 and 74% of the larval ovaries survive the freezing process. 11. Reimplantation of the larval ovaries into larval hosts is decisive for the effective success of the freezing method. However, there is virtually no difference in the subsequent development of either freeze-treated or untreated implants. 12. Transplanted larval ovaries connect to the host oviducts with a certain probability which is related to their genotype. In any case, donor ovaries, freeze-treated or untreated, produce on the average much less offspring than do host ovaries. 13. The suitability of the newly developed freezing technique for the conservation ofDrosophila mutant stocks is discussed.