Efficient Production of Doubled Haploids as Isogenic Line Founders via Double Heat Shock Treatment in Willow Gudgeon (Gnathopogon caerulescens)

Doubled haploids (DHs) are essential founders for breeding via isogenic line production. We determined the optimal timing of the first and second (HST1 and HST2) double heat shock treatment for DH production in the willow gudgeon (Gnathopogon caerulescens). Gynogenesis was induced by fertilisation with UV-irradiated sperm (20 mJ/cm(2)). Each HST was performed at 40.5 degrees C for 1 min. In experiment I, HST1 was applied 23-31 min after the induction of gynogenetic development (incubated at 20 degrees C). HST2 was performed 15 min after HST1 treatment. When HST1 was started 25-26 min after activation, normally hatched larvae appeared relatively frequently (35.6-100%), and most were DHs. In experiment II, HST1 was started 26 min after activation, and HST2 was started 10-25 min after HST1. Starting HST2 10 min after HST1 resulted in 33% more normally hatched larvae than starting 22.5-25 min after. DHs were prevalent among hatched larvae (45.1% and 20.8%, respectively) when performing HST2 10-12.5 min after HST1. Under appropriate HST1 and HST2 timing, gynogens were at the zygote stage, early prophase, and prophase or early prometaphase of the first cell cycle. These results establish the appropriate timing of HST1 and HST2 for the induction of willow gudgeon DHs.