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

Figure 1

Schematic illustration of the experimental procedure, collection of gametes, genetic inactivation of sperm, fertilisation, heat shock treatment (HST), and developed egg collection. (a) Collection of semen from five males. (b) 100-fold dilution of sperm suspension in artificial seminal plasma (ASP). (c) Genetic inactivation of sperm by irradiation with ultraviolet light (UV-sperm). (d) Ripe eggs are stripped by pressing the abdomen of females. They are then divided into three groups (two small groups and one large group). (e) Insemination of eggs with diluted semen (1 mL in Teflon dish) from small groups as intact control (IC). (f) Insemination of eggs from another small group with UV-irradiated sperm as untreated gynogenetic control (GC). (g) Insemination of a large group of eggs with UV-irradiated sperm for experiments involving exposure to HST. (h) Scattering of fertilised eggs into a water tank filled with circulating water (20.0°C) using a Teflon spoon. The eggs then attach to the glass slide and frosted glass plate on the bottom of the tank. (i) A prepositioned frosted glass plate on the bottom of the incubation chamber, to which slide glass is connected with a double clip. (j) Frosted glass plates and glass slides taken out of a water tank. Developed eggs are attached. (k) Collection and fixation of developed eggs for cytological analysis. The glass slides connected to the frosted glass plates are separated and immersed in Bouin’s fixative at 5 s before the start of HST. (l) HST. The first and second treatments are performed by immersing the eggs in water at 40.5°C for 1 min.