Efficient strategies for changing the diapause character of silkworm eggs and for the germline transformation of diapause silkworm strains

To overcome the disadvantages of current silkworm Bombyx mori transgenic technology, such as costly and time-consuming to maintain non-diapause transgenic silkworms, we report here on the development of treatments for the germline transformation of diapause silkworm strains. Our results showed that HCl treatment within 3 h of oviposition was able to prevent the diapause of eggs from Japanese lineage diapause silkworm strains and was also suitable for germline transformation of the same strains. By incubating developing mother eggs from Chinese lineage diapause silkworm strains at 15 degrees C (15 degrees C-IME), we were able to prevent the diapause of their daughter eggs; a similar strategy (15 degrees C-IMES) for the germline transformation of the same strains was that the mother eggs were incubated at 15 degrees C, and the daughter eggs were then microinjected according to the conventional microinjection methods used for non-diapause eggs. By combining temperature and light controls, the improved 15 degrees C-IMES strategy prevented diapause in daughter eggs, and also enabled the germline transformation of both Japanese and Chinese lineage diapause silkworm strains. Although each of the strategies developed here has advantages and disadvantages, we suggest that the 15 degrees C-IMES strategy is a good reference for the establishment of germline transformation technologies of other egg diapause insects. These new strategies for the efficient germline transformation of diapause silkworm strains are likely to improve the practical use of silkworm transgenic lines in sericulture and also highlight silkworm functional genomics research and its modeling.