Preservation of a transgenic strain of the sawfly, Athalia rosae (Hymenoptera) by artificial fertilization using cryopreserved sperm.

Germline transformation using a piggyBac-derived vector is feasible in the sawfly, Athalia rosae. A previously generated transgenic line carrying green fluorescence protein (GFP) genes as reporters was successfully maintained and preserved without consecutive rearing. Sperm taken from males that were frozen directly in liquid nitrogen and stored at -80 degrees C for a year were microinjected into mature unfertilized eggs dissected from female ovaries. A fraction of the sperm-injected eggs was fertilized and developed into diploid females, and all of them expressed GFP. Haploid male progeny from these females segregated into GFP-positive and GFP-negative individuals in a ratio of 1:1 indicating heterozygosity of the parental females. The GFP genes were stably inherited staying at the location where they were originally integrated.