CRYOPRESERVATION OF TOTIPOTENT NUCLEI FROM HONEYBEE (APIS-MELLIFERA) EMBRYOS BY RAPID FREEZING

Here we report a method to cryopreserve totipotent preblastoderm nucle i from honeybee (Apis mellifera) embryos by rapid freezing without the addition of a specific cryoprotectant or other additive. By making fe asible long-term storage of ooplasm biopsied from preblastoderm embryo s, the method could become a key element for the development of an eff icient and practical procedure for cloning and other genetic studies o f honeybees. Donor ooplasm to be cryopreserved was extracted from the anterior pole of 8.0-to 9.0-h preblastoderm embryos with a transplanta tion pipet. The anterior end of the pipet was plugged with a small bal l of cotton fiber, before it was fastened with a tape to a fine iron w ire and plunged into liquid nitrogen. After being stored in liquid nit rogen for at least 24 h, the pipet was thawed rapidly in a water bath at 25 degrees C. The ooplasm was injected at the anterior pole of reci pient embryos (0.5 to 3.5 h) at room temperature and at a relative hum idity >70%. Chimerism was assayed on 3-day larvae by making use of the polymorphic malate dehydrogenase locus and isoelectric focusing. Of 6 18 recipient embryos injected with cryopreserved nuclei, 157 (25.4%) h atched into larvae. Of the 157 larvae, 16 were chimeras carrying bath donor and recipient genotypes and 2 expressed only the donor genotype and thereby were presumed to be embryo clones. The chimeric frequency (11.5%) was 85% of the frequency achieved when using noncryopreserved nuclei and the same transplantation protocol. (C) 1997 Academic Press.